Anti il-36r antibodies

ABSTRACT

The present invention relates to anti-IL-36R binding compounds, in particular new anti-IL-36R antibodies and therapeutic and diagnostic methods and compositions for using the same.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 12, 2012, isnamed 09-0583US.txt and is 146,203 bytes in size.

TECHNICAL FIELD OF THE INVENTION

This invention generally relates to anti-IL-36R antibodies fordiagnostic and therapeutic use. The antibodies can be used inpharmaceutical compositions and kits comprising such compounds. Theantibodies are useful in methods for the treatment of various diseasesor disorders, for example immunological, inflammatory, autoimmune,fibrotic and respiratory diseases in humans.

BACKGROUND OF THE INVENTION

The IL-1 family of cytokines is composed of 11 different ligands,namely, IL-1a (also termed IL-1F1), IL-1β (IL-1F2), IL-1 receptorantagonist (IL-1Ra or IL-1F3), IL-18 (IL-1F4), IL-1F5 to IL-1F10, andIL-1F11 (or IL-33). IL-1a and IL-1β are known to induce pro-inflammatoryactivities on binding to type I IL-1 receptor (IL-1RI) and recruitmentof the common co-receptor IL-1 receptor accessory protein (IL-1RAcP),whereas IL-1Ra acts as a competitive inhibitor of IL-1 binding toIL-1RI, thus exerting anti-inflammatory activity. Numerous studiesreported that IL-18 is a pro-inflammatory cytokine that is an inducer ofIFN-γ, whereas IL-33 was described as an immunoregulatory cytokineinvolved in particular in the control of Th2 responses. New members ofthe IL-1 family, including IL-1F5, IL-1F6, IL-1F8, and IL-1F9, wereidentified through searches in DNA databases for homologs of IL-1. Inhumans and mice, all the genes encoding these cytokines map to less than300 kb of chromosome 2q, where they are flanked by the IL1A, IL1B, andIL1RN genes. IL-1F6, IL-1F8, and IL-1F9 share 21% to 37% amino acidsequence homology with IL-1 and IL-1Ra, whereas IL-1F5 displays 52%amino acid sequence homology with IL-1Ra, suggesting that IL-1F5 mightrepresent an endogenous receptor antagonist.

IL-1F6, IL-1F8, and IL-1F9 bind to IL-1Rrp2, a receptor of the IL-1Rfamily, and use IL-1RAcP as a co-receptor to stimulate intracellularsignals similar to those induced by IL-1, whereas IL-1F5 was shown toinhibit IL-1F9-induced NF-κB activation in Jurkat T cells thatover-express IL-1Rrp2. Like IL-1β, all these IL-1 homologs lack a leaderpeptide and cannot be released through the conventional secretorypathway, although studies suggest that release of IL-1Rrp2 agonists maybe controlled by mechanisms different from those regulating IL-1βsecretion. To acknowledge the specific biologic effects of thesecytokines and to recognize that they all bind to the same receptor, ithas recently been proposed to amend the nomenclature of IL-1 homologs.Thus, IL-1Rrp2 is now termed IL-36R and its ligands are named IL-36α(IL-1F6), IL-36β (IL-1F8), and IL-36γ (IL-1F9). In addition, IL-1F5,which has been shown to exert receptor antagonist activities, has beenrenamed IL-36Ra.

Messenger RNAs for IL-36α, IL-36(3, and IL-36γ are highly expressed inseveral tissues, particularly in internal epithelial tissues, which areexposed to pathogens and in skin. Interestingly, expression of IL-36Raand IL-36α is significantly up-regulated in IL-1β/TNF-α-stimulated humankeratinocytes, and IL-36Ra and IL-36γ mRNA are highly increased inlesional psoriasis skin. Moreover, IL-36γ protein production is enhancedin human keratinocytes after TNF-α and IFN-γ stimulation. ElevatedIL-36α mRNA and protein expression was reported also in chronic kidneydisease.

Transgenic mice overexpressing IL-36α in keratinocytes exhibitinflammatory skin lesions sharing some features with psoriasis. Thisphenotype was more severe when transgenic mice were crossed withIL-36Ra-deficient mice, supporting a regulatory function of IL-36Ra invivo. The inflammatory skin condition in keratinocyte-specific IL-36atransgenic is even more similar to human psoriasis if the mice aretreated with 12-O-tetradecanoylphorbol 13-acetate, resembling the humandisease histologically, molecularly, and in its response totherapeutics. Moreover, human psoriatic lesional skin transplanted ontoimmunodeficient mice is normalized when the mice are treated withanti-IL-36R antibody, arguing that the IL-36 axis is required tomaintain the lesional phenotype in human psoriatic skin. Taken together,these data indicate that IL-36R ligands, including IL-36α, IL-36β, andIL-36γ, exert proinflammatory effects in vitro and in vivo and thatIL-36Ra acts as a natural antagonist, thus mimicking the IL-1/IL-1Rasystem.

There is therefore evidence that IL-36R ligands are involved in a numberof disease conditions, and there is a need for new therapeutic agentstargeting this pathway, in particular for use in the treatment ofinflammatory diseases.

SUMMARY OF THE INVENTION

The present invention addresses the above need by providingbiotherapeutics, in particular antibodies, which bind to IL-36R. In oneaspect, the antibodies of the present invention block IL36ligand-mediated signaling (α, β and/or γ). In one aspect the antibodiesof the present invention are useful, for example for the treatment ofepithelial-mediated inflammation/fibrosis in diseases such as psoriasis,inflammatory bowel disease, scleroderma, COPD, and chronic kidneydisease.

In one aspect, the present invention provides an anti-IL-36R antibodyhaving one or more of the properties below.

In one aspect, an anti-IL-36R antibody of the present invention has highmolecular/cellular binding potency. In one aspect, an anti-IL-36Rantibody of the present invention binds to human IL-36R at a K_(D)<0.1nM. In a further aspect, an anti-IL-36R antibody of the presentinvention, in particular a humanized anti-IL-36R antibody, binds tohuman IL-36R at a K_(D)<50 pM. In one aspect, an anti-IL-36R antibody ofthe present invention binds to IL-36R expressing cells at an EC₉₀<5 nM.

In another aspect, an anti-IL-36R antibody of the present invention hashigh cell-based functional blocking potency. In one aspect, ananti-IL-36R antibody of the present invention blocks all three IL-36Ragonistic ligands (α, β, γ) at an IC₉₀<5 nM, in disease-relevant celllines and primary cells.

In one aspect, an anti-IL-36R antibody of the present invention has themolecular/cellular binding potency and the cell-based functionalblocking potency set forth above.

In a further aspect, an anti-IL-36R antibody of the present inventionhas high selectivity for example greater than 1000-fold selectivityagainst human IL-1R1 or IL-36R negative cell lines. In a further aspect,an anti-IL-36R antibody of the present invention does not bind to humanIL-1R1 or IL-36R negative cell lines.

In embodiment one, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof, which binds to humanIL-36R at a K_(D) equal to or <0.1 nM.

In embodiment two, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodimentone, wherein the said antibody or antigen-binding fragment is amonoclonal antibody or antigen-binding fragment thereof.

In embodiment three, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodiment oneor two, wherein the said antibody or antigen-binding fragment is ahumanized antibody or antigen-binding fragment thereof.

In embodiment four, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodimentthree, which binds to human IL-36R at a K_(D) equal to or <50 pM.

In embodiment five, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to any one ofembodiment one to four, which does not bind to human IL-1R1.

In embodiment six, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodimentone, wherein the antibody or antigen-binding fragment thereof comprises:

a) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 26 (L-CDR1); the amino acid sequence of SEQ ID NO: 35, 102,103, 104, 105 106 or 140 (L-CDR2); the amino acid sequence of SEQ ID NO:44 (L-CDR3); andb) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 53 (H-CDR1); the amino acid sequence of SEQ ID NO: 62, 108,109, 110 or 111 (H-CDR2); the amino acid sequence of SEQ ID NO: 72(H-CDR3).

In embodiment seven, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodimentsix, wherein the antibody or antigen-binding fragment fragment thereofcomprises:

a) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 26 (L-CDR1); the amino acid sequence of SEQ ID NO: 102(L-CDR2); the amino acid sequence of SEQ ID NO: 44 (L-CDR3); andb) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 53 (H-CDR1); the amino acid sequence of SEQ ID NO: 62, 108,109, 110 or 111 (H-CDR2); the amino acid sequence of SEQ ID NO: 72(H-CDR3).

In embodiment eight, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodimentsix, wherein the antibody or antigen-binding fragment fragment thereofcomprises:

a) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 26 (L-CDR1); the amino acid sequence of SEQ ID NO: 103(L-CDR2); the amino acid sequence of SEQ ID NO: 44 (L-CDR3); andb) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 53 (H-CDR1); the amino acid sequence of SEQ ID NO: 62, 108,109, 110 or 111 (H-CDR2); the amino acid sequence of SEQ ID NO: 72(H-CDR3).

In embodiment nine, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodimentsix, wherein the antibody or antigen-binding fragment fragment thereofcomprises:

a) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 26 (L-CDR1); the amino acid sequence of SEQ ID NO: 104(L-CDR2); the amino acid sequence of SEQ ID NO: 44 (L-CDR3); andb) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 53 (H-CDR1); the amino acid sequence of SEQ ID NO: 62, 108,109, 110 or 111 (H-CDR2); the amino acid sequence of SEQ ID NO: 72(H-CDR3).

In embodiment ten, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodimentsix, wherein the antibody or antigen-binding fragment fragment thereofcomprises:

a) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 26 (L-CDR1); the amino acid sequence of SEQ ID NO: 105(L-CDR2); the amino acid sequence of SEQ ID NO: 44 (L-CDR3); andb) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 53 (H-CDR1); the amino acid sequence of SEQ ID NO: 62, 108,109, 110 or 111 (H-CDR2); the amino acid sequence of SEQ ID NO: 72(H-CDR3).

In embodiment eleven, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodimentsix, wherein the antibody or antigen-binding fragment fragment thereofcomprises:

a) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 26 (L-CDR1); the amino acid sequence of SEQ ID NO: 106(L-CDR2); the amino acid sequence of SEQ ID NO: 44 (L-CDR3); andb) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 53 (H-CDR1); the amino acid sequence of SEQ ID NO: 62, 108,109, 110 or 111 (H-CDR2); the amino acid sequence of SEQ ID NO: 72(H-CDR3).

In embodiment twelve, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodimentsix, wherein the antibody or antigen-binding fragment fragment thereofcomprises:

a) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 26 (L-CDR1); the amino acid sequence of SEQ ID NO: 140(L-CDR2); the amino acid sequence of SEQ ID NO: 44 (L-CDR3); andb) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 53 (H-CDR1); the amino acid sequence of SEQ ID NO: 62, 108,109, 110 or 111 (H-CDR2); the amino acid sequence of SEQ ID NO: 72(H-CDR3).

In embodiment thirteen, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodimentone, wherein the antibody or antigen-binding fragment fragment thereofcomprises a light chain variable region comprising the amino acidsequence of any one of SEQ ID NO: 76, 77, 78, 79, 80, 81, 82 or 83; anda heavy chain variable region comprising the amino acid sequence of anyone of SEQ ID NO: 87, 88, 89, 90, 91, 92, 93, 94 or 95.

In embodiment fourteen, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodimentthirteen, wherein the antibody or antigen-binding fragment fragmentthereof comprises a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 77; and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 87; or

a light chain variable region comprising the amino acid sequence of SEQID NO: 77;and a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 88; ora light chain variable region comprising the amino acid sequence of SEQID NO: 77; and a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 89.

In embodiment fifteen, the present invention provides an in the antibodyor antigen-binding fragment fragment thereof comprises a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 80; anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 87; or

a light chain variable region comprising the amino acid sequence of SEQID NO: 80;and a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 88; ora light chain variable region comprising the amino acid sequence of SEQID NO: 80; and a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 89.

In embodiment sixteen, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodimentone, wherein the antibody or antigen-binding fragment fragment thereofcomprises:

a light chain variable region comprising the amino acid sequence of SEQID NO: 27 (L-CDR1); the amino acid sequence of SEQ ID NO: 36 (L-CDR2);the amino acid sequence of SEQ ID NO: 45 (L-CDR3); and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 107(H-CDR1); the amino acid sequence of SEQ ID NO: 63 (H-CDR2); the aminoacid sequence of SEQ ID NO: 73 (H-CDR3); ora light chain variable region comprising the amino acid sequence of SEQID NO: 27 (L-CDR1); the amino acid sequence of SEQ ID NO: 36 (L-CDR2);the amino acid sequence of SEQ ID NO: 45 (L-CDR3); and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 107(H-CDR1); the amino acid sequence of SEQ ID NO: 64 (H-CDR2); the aminoacid sequence of SEQ ID NO: 73 (H-CDR3); ora light chain variable region comprising the amino acid sequence of SEQID NO: 27 (L-CDR1); the amino acid sequence of SEQ ID NO: 36 (L-CDR2);the amino acid sequence of SEQ ID NO: 45 (L-CDR3); and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 54(H-CDR1); the amino acid sequence of SEQ ID NO: 63 or 64 (H-CDR2); theamino acid sequence of SEQ ID NO: 73 (H-CDR3).

In embodiment seventeen, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodimentone, wherein the antibody or antigen-binding fragment fragment thereofcomprises a light chain variable region comprising the amino acidsequence of any one of SEQ ID NO: 84, 85 or 86; and a heavy chainvariable region comprising the amino acid sequence of any one of SEQ IDNO: 96, 97, 98, 99, 100 or 101.

In embodiment eighteen, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodimentseventeen, wherein the antibody or antigen-binding fragment fragmentthereof comprises a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 85; and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 100; or a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 85; anda heavy chain variable region comprising the amino acid sequence of SEQID NO:101.

In embodiment nineteen, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodimentseventeen, wherein the antibody or antigen-binding fragment fragmentthereof comprises a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 86; and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 100; or a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 86; anda heavy chain variable region comprising the amino acid sequence of SEQID NO:101.

In embodiment twenty, the present invention provides an anti-IL-36Rantibody, wherein the antibody comprises a light chain comprising theamino acid sequence of any one of SEQ ID NO: 114, 115, 116, 117, 118,119, 120 or 121; and a heavy chain comprising the amino acid sequence ofany one of SEQ ID NO: 125, 126, 127, 128, 129, 130, 131, 132 or 133.

In embodiment twenty one, the present invention provides an anti-IL-36Rantibody according to embodiment twenty, wherein the antibody comprisesa light chain comprising the amino acid sequence of SEQ ID NO: 115; anda heavy chain comprising the amino acid sequence of SEQ ID NO: 125.

In embodiment twenty two, the present invention provides an anti-IL-36Rantibody according to embodiment twenty, wherein the antibody comprisesa light chain comprising the amino acid sequence of SEQ ID NO: 115; anda heavy chain comprising the amino acid sequence of SEQ ID NO: 126.

In embodiment twenty three, the present invention provides ananti-IL-36R antibody according to embodiment twenty, wherein theantibody comprises a light chain comprising the amino acid sequence ofSEQ ID NO: 115; and a heavy chain comprising the amino acid sequence ofSEQ ID NO: 127.

In embodiment twenty four, the present invention provides an anti-IL-36Rantibody according to embodiment twenty, wherein the antibody comprisesa light chain comprising the amino acid sequence of SEQ ID NO: 118; anda heavy chain comprising the amino acid sequence of SEQ ID NO: 125.

In embodiment twenty five, the present invention provides an anti-IL-36Rantibody according to embodiment twenty, wherein the antibody comprisesa light chain comprising the amino acid sequence of SEQ ID NO: 118; anda heavy chain comprising the amino acid sequence of SEQ ID NO: 126.

In embodiment twenty six, the present invention provides an anti-IL-36Rantibody according to embodiment twenty, wherein the antibody comprisesa light chain comprising the amino acid sequence of SEQ ID NO: 118; anda heavy chain comprising the amino acid sequence of SEQ ID NO: 127.

In embodiment twenty seven, the present invention provides ananti-IL-36R antibody, wherein the antibody comprises a light chaincomprising the amino acid sequence of any one of SEQ ID NO: 122, 123 or124; and a heavy chain comprising the amino acid sequence of any one ofSEQ ID NO: 134, 135, 136, 137, 138 or 139.

In embodiment twenty eight, the present invention provides ananti-IL-36R antibody according to embodiment twenty seven, wherein theantibody comprises a light chain comprising the amino acid sequence ofSEQ ID NO: 123; and a heavy chain comprising the amino acid sequence ofSEQ ID NO: 138.

In embodiment twenty nine, the present invention provides an anti-IL-36Rantibody according to embodiment twenty seven, wherein the antibodycomprises a light chain comprising the amino acid sequence of SEQ ID NO:123; and a heavy chain comprising the amino acid sequence of SEQ ID NO:139.

In embodiment thirty, the present invention provides an anti-IL-36Rantibody according to twenty seven, wherein the antibody comprises alight chain comprising the amino acid sequence of SEQ ID NO: 124; and aheavy chain comprising the amino acid sequence of SEQ ID NO: 138.

In embodiment thirty one, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodimentone, wherein the antibody or antigen-binding fragment fragment thereofcomprises:

a) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 26 (L-CDR1); the amino acid sequence of SEQ ID NO: 103(L-CDR2); the amino acid sequence of SEQ ID NO: 44 (L-CDR3); andb) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 53 (H-CDR1); the amino acid sequence of SEQ ID NO: 62(H-CDR2); the amino acid sequence of SEQ ID NO: 72 (H-CDR3).

In embodiment thirty two, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodimentone, wherein the antibody or antigen-binding fragment fragment thereofcomprises:

a) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 26 (L-CDR1); the amino acid sequence of SEQ ID NO:104(L-CDR2); the amino acid sequence of SEQ ID NO: 44 (L-CDR3); andb) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 53 (H-CDR1); the amino acid sequence of SEQ ID NO: 62(H-CDR2); the amino acid sequence of SEQ ID NO: 72 (H-CDR3).

In embodiment thirty three, the present invention provides ananti-IL-36R antibody or antigen-binding fragment thereof according toembodiment one, wherein the antibody or antigen-binding fragmentfragment thereof comprises:

a) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 27 (L-CDR1); the amino acid sequence of SEQ ID NO:36(L-CDR2); the amino acid sequence of SEQ ID NO: 45 (L-CDR3); andb) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 107 (H-CDR1); the amino acid sequence of SEQ ID NO: 63(H-CDR2); the amino acid sequence of SEQ ID NO: 73 (H-CDR3).

In embodiment thirty four, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof according to embodimentone, wherein the antibody or antigen-binding fragment fragment thereofcomprises:

a) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 27 (L-CDR1); the amino acid sequence of SEQ ID NO:36(L-CDR2); the amino acid sequence of SEQ ID NO: 45 (L-CDR3); andb) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 107 (H-CDR1); the amino acid sequence of SEQ ID NO: 64(H-CDR2); the amino acid sequence of SEQ ID NO: 73 (H-CDR3).

In one embodiment, an antibody or antigen-binding fragment thereofaccording to any one of embodiments one to thirty-four is a monoclonalantibody. In one embodiment, an antibody or antigen-binding fragmentthereof according to any one of embodiments one to thirty-four is ahumanized antibody. In one embodiment, an antibody or antigen-bindingfragment thereof according to any one of embodiments one to thirty-fouris a monoclonal humanized antibody.

In embodiment thirty five, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof, wherein the antibody orantigen-binding fragment fragment thereof comprises:

a light chain variable region comprising the amino acid sequence of SEQID NO: 21 (L-CDR1); the amino acid sequence of SEQ ID NO: 30 (L-CDR2);the amino acid sequence of SEQ ID NO: 39 (L-CDR3); and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 48(H-CDR1); the amino acid sequence of SEQ ID NO: 57 (H-CDR2); the aminoacid sequence of SEQ ID NO: 67 (H-CDR3); ora light chain variable region comprising the amino acid sequence of SEQID NO: 22 (L-CDR1); the amino acid sequence of SEQ ID NO: 31 (L-CDR2);the amino acid sequence of SEQ ID NO: 40 (L-CDR3); and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 49(H-CDR1); the amino acid sequence of SEQ ID NO: 58 (H-CDR2); the aminoacid sequence of SEQ ID NO: 68 (H-CDR3); ora light chain variable region comprising the amino acid sequence of SEQID NO: 23 (L-CDR1); the amino acid sequence of SEQ ID NO: 32 (L-CDR2);the amino acid sequence of SEQ ID NO: 41 (L-CDR3); and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 50(H-CDR1); the amino acid sequence of SEQ ID NO: 59 (H-CDR2); the aminoacid sequence of SEQ ID NO: 69 (H-CDR3); ora light chain variable region comprising the amino acid sequence of SEQID NO: 24 (L-CDR1); the amino acid sequence of SEQ ID NO: 33 (L-CDR2);the amino acid sequence of SEQ ID NO: 42 (L-CDR3); and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 51(H-CDR1); the amino acid sequence of SEQ ID NO: 60 (H-CDR2); the aminoacid sequence of SEQ ID NO: 70 (H-CDR3); ora light chain variable region comprising the amino acid sequence of SEQID NO: 25 (L-CDR1); the amino acid sequence of SEQ ID NO: 34 (L-CDR2);the amino acid sequence of SEQ ID NO: 43 (L-CDR3); and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 52(H-CDR1); the amino acid sequence of SEQ ID NO: 61 (H-CDR2); the aminoacid sequence of SEQ ID NO: 71 (H-CDR3); ora light chain variable region comprising the amino acid sequence of SEQID NO: 26 (L-CDR1); the amino acid sequence of SEQ ID NO: 35 (L-CDR2);the amino acid sequence of SEQ ID NO: 44 (L-CDR3); and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 53(H-CDR1); the amino acid sequence of SEQ ID NO: 62 (H-CDR2); the aminoacid sequence of SEQ ID NO: 72 (H-CDR3); ora light chain variable region comprising the amino acid sequence of SEQID NO: 27 (L-CDR1); the amino acid sequence of SEQ ID NO: 36 (L-CDR2);the amino acid sequence of SEQ ID NO: 45 (L-CDR3); and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 54(H-CDR1); the amino acid sequence of SEQ ID NO: 63 (H-CDR2); the aminoacid sequence of SEQ ID NO: 73 (H-CDR3); ora light chain variable region comprising the amino acid sequence of SEQID NO: 27 (L-CDR1); the amino acid sequence of SEQ ID NO: 36 (L-CDR2);the amino acid sequence of SEQ ID NO: 45 (L-CDR3); and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 54(H-CDR1); the amino acid sequence of SEQ ID NO: 64 (H-CDR2); the aminoacid sequence of SEQ ID NO: 73 (H-CDR3); or a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 28 (L-CDR1); theamino acid sequence of SEQ ID NO: 37 (L-CDR2); the amino acid sequenceof SEQ ID NO: 46 (L-CDR3); and a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 55 (H-CDR1); the amino acidsequence of SEQ ID NO: 65 (H-CDR2); the amino acid sequence of SEQ IDNO: 74 (H-CDR3); ora light chain variable region comprising the amino acid sequence of SEQID NO: 29 (L-CDR1); the amino acid sequence of SEQ ID NO: 38 (L-CDR2);the amino acid sequence of SEQ ID NO: 47 (L-CDR3); and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 56(H-CDR1); the amino acid sequence of SEQ ID NO: 66 (H-CDR2); the aminoacid sequence of SEQ ID NO: 75 (H-CDR3).

In embodiment thirty six, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof, wherein the antibody orantigen-binding fragment fragment thereof comprises a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 1; anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 11; or

a light chain variable region comprising the amino acid sequence of SEQID NO: 2; anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 12; ora light chain variable region comprising the amino acid sequence of SEQID NO: 3; anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 13; ora light chain variable region comprising the amino acid sequence of SEQID NO: 4; anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 14; ora light chain variable region comprising the amino acid sequence of SEQID NO: 5; anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 15; ora light chain variable region comprising the amino acid sequence of SEQID NO: 6; anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 16; ora light chain variable region comprising the amino acid sequence of SEQID NO: 7; anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 17; ora light chain variable region comprising the amino acid sequence of SEQID NO: 8; anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 18; ora light chain variable region comprising the amino acid sequence of SEQID NO: 9; anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 19; ora light chain variable region comprising the amino acid sequence of SEQID NO: 10;and a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 20.

In a further embodiment thirty seven, the present invention provides apharmaceutical composition comprising an antibody or antigen-bindingfragment according to any one of the previous embodiments and apharmaceutically acceptable carrier.

In a further embodiment thirty eight the present invention provides anantibody or antigen-binding fragment or pharmaceutical compositionaccording to any one of the previous embodiments, for use in medicine.

In a further embodiment thirty nine the present invention provides anantibody or antigen-binding fragment or pharmaceutical compositionaccording to any one of the embodiments 1-37, wherein the use is thetreatment of an inflammatory disease, of an autoimmune disease, of arespiratory disease, of a metabolic disorder, of an epithelial mediatedinflammatory disorder, fibrosis or of cancer.

In a further embodiment forty the present invention provides an antibodyor antigen-binding fragment or pharmaceutical composition according toany one of the embodiments 1-37, wherein the use is for the treatment ofpsoriasis, inflammatory bowel disease, psoriatic arthritis, multiplesclerosis, rheumatoid arthritis, COPD, chronic asthma or ankylosingspondylitis.

In a further embodiment forty one, the present invention provides anantibody or antigen-binding fragment or pharmaceutical compositionaccording to any one of the embodiments 1-37, wherein the use is for thetreatment of inflammatory bowel disease.

In a still further embodiment forty two, the present invention providesan antibody or antigen-binding fragment or pharmaceutical compositionaccording to embodiment 41, wherein the disease is Crohns disease.

In another embodiment forty three, the present invention provides amethod of treating a disease comprising administering the antibody orantigen-binding fragment or pharmaceutical composition according to anyone of the embodiments 1-37, to a patient in need thereof, wherein thedisease is selected from an inflammatory disease, an autoimmune disease,a respiratory disease, a metabolic disorder, an epithelial mediatedinflammatory disorder, fibrosis and cancer.

In another embodiment forty four, the present invention provides amethod according to embodiment 43 wherein the disease is selected frompsoriasis, inflammatory bowel disease, psoriatic arthritis, multiplesclerosis, rheumatoid arthritis, COPD, chronic asthma and ankylosingspondylitis.

In a still further embodiment forty five, the present invention providesa method for treating Crohns disease.

Further embodiments of the invention encompass:

-   -   An isolated polynucleotide comprising a sequence encoding an        anti-IL-36R antibody or antigen-binding fragment according to        the invention, preferably a DNA or RNA sequence;    -   an isolated polynucleotide according to the invention, encoding        a sequence as defined by one or more of SEQ ID NOs. 1 to 140;    -   a vector comprising a polynucleotide according to the invention,        preferably an expression vector, more preferred a vector        comprising the polynucleotide according to the invention in        functional association with an expression control sequence;    -   a host cell comprising a polynucleotide according to the        invention and/or a vector according to the invention;    -   a method for the production of an anti-IL-36R antibody or        antigen-binding fragment according to the invention, preferably        a recombinant production method comprising the use of a        polynucleotide according to the invention, and/or of a vector        according to the invention and/or of a host cell according to        the invention;    -   such a method preferably comprises the steps (a) cultivating the        host cell under conditions allowing the expression of the        anti-IL-36R antibody or antigen-binding fragment and (b)        recovering the anti-IL-36R antibody or antigen-binding fragment;    -   a diagnostic kit or diagnostic method comprising an anti-IL-36R        antibody or antigen-binding fragment according to the invention,        or the use thereof;    -   a Diagnostic kit or diagnostic method according the invention,        for the diagnosis of an inflammatory disease, an autoimmune        disease, a respiratory disease, a metabolic disorder, an        epithelial mediated inflammatory disorder, fibrosis, cancer,        psoriasis, inflammatory bowel disease, psoriatic arthritis,        multiple sclerosis, rheumatoid arthritis, COPD, chronic asthma,        ankylosing spondylitis, or Crohns disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: IL-36 antagonist ligands (IL-36RA/IL1F5, IL-38/ILF10) inhibitthe signaling cascade.

FIG. 2: Gene chip analyses demonstrate IL-36R ligands are upregulated inpsoriatic skin (IL-36 RA, IL-36 α and IL-36 γ).

FIG. 3: Expression profile using human skin sections. Formalin-fixedparaffin embedded with antibody titrations using antibody 33D10

FIG. 4: Method to Evaluate Epidermal thickness of human skin sections

DESCRIPTION OF THE INVENTION

This invention relates to anti-IL-36R antibodies. In one aspect, theantibodies of the present invention are for diagnostic and therapeuticuse, for example in humans.

The present invention provides antibodies that bind to IL-36R, inparticular human IL-36R. The present invention also relates to humanizedantibodies that bind IL-36R. In specific embodiments, the sequence ofthese humanized antibodies has been identified based on the sequences ofcertain lead mouse antibodies.

Without wishing to be bound by this theory it is believed thatanti-IL-36R antibodies or antigen-binding fragments thereof bind tohuman IL-36R and thus interfere with the binding of IL-36 agonists, andin doing so block at least partially the signaling cascade from theIL-36R to inflammatory mediators. This is illustrated by FIG. 1.

In one aspect, the antibodies of the present invention are for use inmodels of human disease. IL-36R is also known as IL-1RL2 and IL-1Rrp2.It has been reported that agonistic IL-36 ligands (α, β, or γ) initiatethe signaling cascade by engaging the IL-36 receptor which then forms aheterodimer with the IL-1 receptor accessory protein (IL-1RAcP). IL-36antagonist ligands (IL-36RA/IL1F5, IL-38/ILF10) inhibit the signalingcascade (see FIG. 1).

In one aspect, the present invention provides an anti-IL-36R antibodyhaving one or more of the properties below.

In one aspect, an anti-IL-36R antibody of the present invention has highmolecular/cellular binding potency. In one aspect, an anti-IL-36Rantibody of the present invention binds to human IL-36R at a K_(D)<0.1nM. In a further aspect, an anti-IL-36R antibody of the presentinvention, in particular a humanized anti-IL-36R antibody, binds tohuman IL-36R at a K_(D)<50 pM. In one aspect, an anti-IL-36R antibody ofthe present invention binds to IL-36R expressing cells at an EC₉₀<5 nM.

In another aspect, an anti-IL-36R antibody of the present invention hashigh cell-based functional blocking potency. In one aspect, ananti-IL-36R antibody of the present invention blocks all three IL-36Ragonistic ligands (α, β, γ) at an IC₉₀<5 nM, in disease-relevant celllines and primary cells.

In one aspect, an anti-IL-36R antibody of the present invention has themolecular/cellular binding potency and the cell-based functionalblocking potency set forth above.

In one aspect, an anti-IL-36R antibody of the present invention is ahumanized antibody. In one aspect, an anti-IL-36R antibody of thepresent invention is a monoclonal antibody. In one aspect, ananti-IL-36R antibody of the present invention is a full length antibody.In one aspect, an anti-IL-36R antibody of the present invention is ahumanized monoclonal antibody, for example a full length humanizedmonoclonal antibody.

An antibody or antigen-binding fragment thereof of the present inventionrecognizes specific “IL-36R antigen epitope” or “IL-36R epitope”. Asused herein these terms refer to a molecule (e.g., a peptide) or afragment of a molecule capable of immunoreactivity with an anti-IL-36Rantibody.

The epitopes are most commonly proteins, short oligopeptides,oligopeptide mimics (i.e., organic compounds that mimic antibody bindingproperties of the IL-36R antigen), or combinations thereof. The minimumsize of a peptide or polypeptide epitope for an antibody is thought tobe about four to five amino acids. Peptide or polypeptide epitopescontain for example at least seven amino acids or for example at leastnine amino acids or for example between about 15 to about 20 aminoacids. Since an antibody can recognize an antigenic peptide orpolypeptide in its tertiary form, the amino acids comprising an epitopeneed not be contiguous, and in some cases, may not even be on the samepeptide chain. Epitopes may be determined by various techniques known inthe art, such as X-ray crystallography, Hydrogen/Deuterium Exchange MassSpectrometry (HXMS), site-directed mutagenesis, alanine scanningmutagenesis, and peptide screening methods.

The generalized structure of antibodies or immunoglobulin is well knownto those of skill in the art. These molecules are heterotetramericglycoproteins, typically of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains and aretypically referred to as full length antibodies. Each light chain iscovalently linked to a heavy chain by one disulfide bond to form aheterodimer, and the heterotrameric molecule is formed through acovalent disulfide linkage between the two identical heavy chains of theheterodimers. Although the light and heavy chains are linked together byone disulfide bond, the number of disulfide linkages between the twoheavy chains varies by immunoglobulin isotype. Each heavy and lightchain also has regularly spaced intrachain disulfide bridges. Each heavychain has at the amino-terminus a variable domain (V_(H)), followed bythree or four constant domains (C_(H1), C_(H2), C_(H3), and C_(H4)), aswell as a hinge region between C_(H1) and C_(H2). Each light chain hastwo domains, an amino-terminal variable domain (V_(L)) and acarboxy-terminal constant domain (C_(L)). The V_(L) domain associatesnon-covalently with the V_(H) domain, whereas the C_(L) domain iscommonly covalently linked to the C_(H1) domain via a disulfide bond.Particular amino acid residues are believed to form an interface betweenthe light and heavy chain variable domains (Chothia et al., 1985, J.Mol. Biol. 186:651-663). Variable domains are also referred herein asvariable regions.

Certain domains within the variable domains differ extensively betweendifferent antibodies i.e., are “hypervariable.” These hypervariabledomains contain residues that are directly involved in the binding andspecificity of each particular antibody for its specific antigenicdeterminant. Hypervariability, both in the light chain and the heavychain variable domains, is concentrated in three segments known ascomplementarity determining regions (CDRs) or hypervariable loops(HVLs). CDRs are defined by sequence comparison in Kabat et al., 1991,In: Sequences of Proteins of Immunological Interest, 5^(th) Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., whereasHVLs (also referred herein as CDRs) are structurally defined accordingto the three-dimensional structure of the variable domain, as describedby Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917. These two methodsresult in slightly different identifications of a CDR. As defined byKabat, CDR-L1 is positioned at about residues 24-34, CDR-L2, at aboutresidues 50-56, and CDR-L3, at about residues 89-97 in the light chainvariable domain; CDR-H1 is positioned at about residues 31-35, CDR-H2 atabout residues 50-65, and CDR-H3 at about residues 95-102 in the heavychain variable domain. The exact residue numbers that encompass aparticular CDR will vary depending on the sequence and size of the CDR.Those skilled in the art can routinely determine which residues comprisea particular CDR given the variable region amino acid sequence of theantibody. The CDR1, CDR2, CDR3 of the heavy and light chains thereforedefine the unique and functional properties specific for a givenantibody.

The three CDRs within each of the heavy and light chains are separatedby framework regions (FR), which contain sequences that tend to be lessvariable. From the amino terminus to the carboxy terminus of the heavyand light chain variable domains, the FRs and CDRs are arranged in theorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The largely β-sheetconfiguration of the FRs brings the CDRs within each of the chains intoclose proximity to each other as well as to the CDRs from the otherchain. The resulting conformation contributes to the antigen bindingsite (see Kabat et al., 1991, NIH Publ. No. 91-3242, Vol. I, pages647-669), although not all CDR residues are necessarily directlyinvolved in antigen binding.

FR residues and Ig constant domains are not directly involved in antigenbinding, but contribute to antigen binding and/or mediate antibodyeffector function. Some FR residues are thought to have a significanteffect on antigen binding in at least three ways: by noncovalentlybinding directly to an epitope, by interacting with one or more CDRresidues, and by affecting the interface between the heavy and lightchains. The constant domains are not directly involved in antigenbinding but mediate various Ig effector functions, such as participationof the antibody in antibody dependent cellular cytotoxicity (ADCC),complement dependent cytotoxicity (CDC) and antibody dependent cellularphagocytosis (ADCP).

The light chains of vertebrate immunoglobulins are assigned to one oftwo clearly distinct classes, kappa (κ) and lambda (λ), based on theamino acid sequence of the constant domain. By comparison, the heavychains of mammalian immunoglobulins are assigned to one of five majorclasses, according to the sequence of the constant domains: IgA, IgD,IgE, IgG, and IgM. IgG and IgA are further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavychain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of the classes of nativeimmunoglobulins are well known.

The terms, “antibody”, “anti-IL-36R antibody”, “humanized anti-IL-36Rantibody”, “humanized anti-IL-36R epitope antibody”, and “varianthumanized anti-IL-36R epitope antibody” specifically encompassmonoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments such as variable domains and otherportions of antibodies that exhibit a desired biological activity, e.g.,IL-36R binding. The term “monoclonal antibody” (mAb) refers to anantibody that is highly specific, being directed against a singleantigenic determinant, an “epitope”. Therefore, the modifier“monoclonal” is indicative of antibodies directed to the identicalepitope and is not to be construed as requiring production of theantibody by any particular method. It should be understood thatmonoclonal antibodies can be made by any technique or methodology knownin the art; including e.g., the hybridoma method (Kohler et al., 1975,Nature 256:495), or recombinant DNA methods known in the art (see, e.g.,U.S. Pat. No. 4,816,567), or methods of isolation of monoclonalrecombinantly produced using phage antibody libraries, using techniquesdescribed in Clackson et al., 1991, Nature 352: 624-628, and Marks etal., 1991, J. Mol. Biol. 222: 581-597.

The term “monomer” refers to a homogenous form of an antibody. Forexample, for a full-length antibody, monomer means a monomeric antibodyhaving two identical heavy chains and two identical light chains.

Chimeric antibodies consist of the heavy and light chain variableregions of an antibody from one species (e.g., a non-human mammal suchas a mouse) and the heavy and light chain constant regions of anotherspecies (e.g., human) antibody and can be obtained by linking the DNAsequences encoding the variable regions of the antibody from the firstspecies (e.g., mouse) to the DNA sequences for the constant regions ofthe antibody from the second (e.g. human) species and transforming ahost with an expression vector containing the linked sequences to allowit to produce a chimeric antibody. Alternatively, the chimeric antibodyalso could be one in which one or more regions or domains of the heavyand/or light chain is identical with, homologous to, or a variant of thecorresponding sequence in a monoclonal antibody from anotherimmunoglobulin class or isotype, or from a consensus or germlinesequence. Chimeric antibodies can include fragments of such antibodies,provided that the antibody fragment exhibits the desired biologicalactivity of its parent antibody, for example binding to the same epitope(see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc.Natl. Acad. Sci. USA 81: 6851-6855).

The terms, “antibody fragment”, “anti-IL-36R antibody fragment”,“anti-IL-36R epitope antibody fragment”, “humanized anti-IL-36R antibodyfragment”, “humanized anti-IL-36R epitope antibody fragment”, “varianthumanized anti-IL-36R epitope antibody fragment” refer to a portion of afull length anti-IL-36R antibody, in which a variable region or afunctional capability is retained, for example, specific IL-36R epitopebinding. Examples of antibody fragments include, but are not limited to,a Fab, Fab′, F(ab′)₂, Fd, Fv, scFv and scFv-Fc fragment, a diabody, alinear antibody, a single-chain antibody, a minibody, a diabody formedfrom antibody fragments, and multispecific antibodies formed fromantibody fragments.

Full length antibodies can be treated with enzymes such as papain orpepsin to generate useful antibody fragments. Papain digestion is usedto produces two identical antigen-binding antibody fragments called“Fab” fragments, each with a single antigen-binding site, and a residual“Fc” fragment. The Fab fragment also contains the constant domain of thelight chain and the C_(H1) domain of the heavy chain. Pepsin treatmentyields a F(ab′)₂ fragment that has two antigen-binding sites and isstill capable of crosslinking antigen.

Fab′ fragments differ from Fab fragments by the presence of additionalresidues including one or more cysteines from the antibody hinge regionat the C-terminus of the C_(H1) domain. F(ab′)₂ antibody fragments arepairs of Fab′ fragments linked by cysteine residues in the hinge region.Other chemical couplings of antibody fragments are also known.

“Fv” fragment contains a complete antigen-recognition and binding siteconsisting of a dimer of one heavy and one light chain variable domainin tight, non-covalent association. In this configuration, the threeCDRs of each variable domain interact to define an antigen-biding siteon the surface of the V_(H)-V_(L) dimer. Collectively, the six CDRsconfer antigen-binding specificity to the antibody.

A “single-chain Fv” or “scFv” antibody fragment is a single chain Fvvariant comprising the V_(H) and V_(L) domains of an antibody where thedomains are present in a single polypeptide chain. The single chain Fvis capable of recognizing and binding antigen. The scFv polypeptide mayoptionally also contain a polypeptide linker positioned between theV_(H) and V_(L) domains in order to facilitate formation of a desiredthree-dimensional structure for antigen binding by the scFv (see, e.g.,Pluckthun, 1994, In The Pharmacology of monoclonal Antibodies, Vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315).

A “diabody” refers to small antibody fragments with two antigen-bindingsites, which fragments comprise a heavy chain variable domain (V.sub.H)connected to a light chain variable domain (V.sub.L) in the samepolypeptide chain (V.sub.H-V.sub.L or V.sub.L-V.sub.H). Diabodies aredescribed more fully in, e.g., Holliger et al. (1993) Proc. Natl. Acad.Sci. USA 90: 6444-6448.

Other recognized antibody fragments include those that comprise a pairof tandem Fd segments (V_(H)-C_(H1)-V_(H)-C_(H1)) to form a pair ofantigen binding regions. These “linear antibodies” can be bispecific ormonospecific as described in, for example, Zapata et al. 1995, ProteinEng. 8(10):1057-1062.

A “humanized antibody” or a “humanized antibody fragment” is a specifictype of chimeric antibody which includes an immunoglobulin amino acidsequence variant, or fragment thereof, which is capable of binding to apredetermined antigen and which, comprises one or more FRs havingsubstantially the amino acid sequence of a human immunoglobulin and oneor more CDRs having substantially the amino acid sequence of a non-humanimmunoglobulin. This non-human amino acid sequence often referred to asan “import” sequence is typically taken from an “import” antibodydomain, particularly a variable domain. In general, a humanized antibodyincludes at least the CDRs or HVLs of a non-human antibody, insertedbetween the FRs of a human heavy or light chain variable domain. Thepresent invention describes specific humanized anti-IL-36R antibodieswhich contain CDRs derived from the mouse monoclonal antibodies orhumanized CDRs inserted between the FRs of human germline sequence heavyand light chain variable domains. It will be understood that certainmouse FR residues may be important to the function of the humanizedantibodies and therefore certain of the human germline sequence heavyand light chain variable domains residues are modified to be the same asthose of the corresponding mouse sequence.

In another aspect, a humanized anti-IL-36R antibody comprisessubstantially all of at least one, and typically two, variable domains(such as contained, for example, in Fab, Fab′, F(ab′)2, Fabc, and Fvfragments) in which all, or substantially all, of the CDRs correspond tothose of a non-human immunoglobulin, and specifically herein, all of theCDRs are mouse or humanized sequences as detailed herein below and all,or substantially all, of the FRs are those of a human immunoglobulinconsensus or germline sequence. In another aspect, a humanizedanti-IL-36R antibody also includes at least a portion of animmunoglobulin Fc region, typically that of a human immunoglobulin.Ordinarily, the antibody will contain both the light chain as well as atleast the variable domain of a heavy chain. The antibody also mayinclude one or more of the C_(H1), hinge, C_(H2), C_(H3), and/or C_(H4)regions of the heavy chain, as appropriate.

A humanized anti-IL-36r antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂. For example, theconstant domain can be a complement fixing constant domain where it isdesired that the humanized antibody exhibit cytotoxic activity, and theisotype is typically IgG₁. Where such cytotoxic activity is notdesirable, the constant domain may be of another isotype, e.g., IgG₂. Analternative humanized anti-IL-36R antibody can comprise sequences frommore than one immunoglobulin class or isotype, and selecting particularconstant domains to optimize desired effector functions is within theordinary skill in the art. In specific embodiments, the presentinvention provides antibodies that are IgG1 antibodies and moreparticularly, are IgG1 antibodies in which there is a knock-out ofeffector functions.

The FRs and CDRs, or HVLs, of a humanized anti-IL-36R antibody need notcorrespond precisely to the parental sequences. For example, one or moreresidues in the import CDR, or HVL, or the consensus or germline FRsequence may be altered (e.g., mutagenized) by substitution, insertionor deletion such that the resulting amino acid residue is no longeridentical to the original residue in the corresponding position ineither parental sequence but the antibody nevertheless retains thefunction of binding to IL-36R. Such alteration typically will not beextensive and will be conservative alterations. Usually, at least 75% ofthe humanized antibody residues will correspond to those of the parentalconsensus or germline FR and import CDR sequences, more often at least90%, and most frequently greater than 95%, or greater than 98% orgreater than 99%.

Immunoglobulin residues that affect the interface between heavy andlight chain variable regions (“the V_(L)-V_(H) interface”) are thosethat affect the proximity or orientation of the two chains with respectto one another. Certain residues that may be involved in interchaininteractions include V_(L) residues 34, 36, 38, 44, 46, 87, 89, 91, 96,and 98 and V_(H) residues 35, 37, 39, 45, 47, 91, 93, 95, 100, and 103(utilizing the numbering system set forth in Kabat et al., Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md., 1987)). U.S. Pat. No. 6,407,213 also discusses thatresidues such as V_(L) residues 43 and 85, and V_(H) residues 43 and 60also may be involved in this interaction. While these residues areindicated for human IgG only, they are applicable across species.Important antibody residues that are reasonably expected to be involvedin interchain interactions are selected for substitution into theconsensus sequence.

The terms “consensus sequence” and “consensus antibody” refer to anamino acid sequence which comprises the most frequently occurring aminoacid residue at each location in all immunoglobulins of any particularclass, isotype, or subunit structure, e.g., a human immunoglobulinvariable domain. The consensus sequence may be based on immunoglobulinsof a particular species or of many species. A “consensus” sequence,structure, or antibody is understood to encompass a consensus humansequence as described in certain embodiments, and to refer to an aminoacid sequence which comprises the most frequently occurring amino acidresidues at each location in all human immunoglobulins of any particularclass, isotype, or subunit structure. Thus, the consensus sequencecontains an amino acid sequence having at each position an amino acidthat is present in one or more known immunoglobulins, but which may notexactly duplicate the entire amino acid sequence of any singleimmunoglobulin. The variable region consensus sequence is not obtainedfrom any naturally produced antibody or immunoglobulin. Kabat et al.,1991, Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., andvariants thereof.

Human germline sequences are found naturally in the human population. Acombination of those germline genes generates antibody diversity.Germline antibody sequences for the light chain of the antibody comefrom conserved human germline kappa or lambda v-genes and j-genes.Similarly the heavy chain sequences come from germline v-, d- andj-genes (LeFranc, M-P, and LeFranc, G, “The Immunoglobulin Facts Book”Academic Press, 2001).

As used herein, “variant”, “anti-IL-36R variant”, “humanized anti-IL-36Rvariant”, or “variant humanized anti-IL-36R” each refers to a humanizedanti-IL-36R antibody having at least a light chain variable murine CDR.Variants include those having one or more amino acid changes in one orboth light chain or heavy chain variable domains, provided that theamino acid change does not substantially impair binding of the antibodyto IL-36R.

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of the antibody's natural environment are thosematerials that may interfere with diagnostic or therapeutic uses of theantibody, and can be enzymes, hormones, or other proteinaceous ornonproteinaceous solutes. In one aspect, the antibody will be purifiedto at least greater than 95% isolation by weight of antibody.

An isolated antibody includes an antibody in situ within recombinantcells in which it is produced, since at least one component of theantibody's natural environment will not be present. Ordinarily however,an isolated antibody will be prepared by at least one purification stepin which the recombinant cellular material is removed.

The term “antibody performance” refers to factors that contribute toantibody recognition of antigen or the effectiveness of an antibody invivo. Changes in the amino acid sequence of an antibody can affectantibody properties such as folding, and can influence physical factorssuch as initial rate of antibody binding to antigen (k_(a)),dissociation constant of the antibody from antigen (k_(d)), affinityconstant of the antibody for the antigen (Kd), conformation of theantibody, protein stability, and half life of the antibody.

The term “epitope tagged” when used herein, refers to an anti-IL-36Rantibody fused to an “epitope tag”. An “epitope tag” is a polypeptidehaving a sufficient number of amino acids to provide an epitope forantibody production, yet is designed such that it does not interferewith the desired activity of the humanized anti-IL-36R antibody. Theepitope tag is usually sufficiently unique such that an antibody raisedagainst the epitope tag does not substantially cross-react with otherepitopes. Suitable tag polypeptides generally contain at least 6 aminoacid residues and usually contain about 8 to 50 amino acid residues, orabout 9 to 30 residues. Examples of epitope tags and the antibody thatbinds the epitope include the flu HA tag polypeptide and its antibody12CA5 (Field et al., 1988 Mol. Cell. Biol. 8: 2159-2165; c-myc tag and8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., 1985,Mol. Cell. Biol. 5(12):3610-3616; and Herpes simplex virus glycoproteinD (gD) tag and its antibody (Paborsky et al. 1990, Protein Engineering3(6): 547-553). In certain embodiments, the epitope tag is a “salvagereceptor binding epitope”. As used herein, the term “salvage receptorbinding epitope” refers to an epitope of the Fc region of an IgGmolecule (such as IgG₁, IgG₂, IgG₃, or IgG₄) that is responsible forincreasing the in vivo serum half-life of the IgG molecule.

In some embodiments, the antibodies of the present invention may beconjugated to a cytotoxic agent. This is any substance that inhibits orprevents the function of cells and/or causes destruction of cells. Theterm is intended to include radioactive isotopes (such as I¹³¹, I¹²⁵,Y⁹⁰, and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant, or animalorigin, and fragments thereof. Such cytotoxic agents can be coupled tothe humanized antibodies of the present invention using standardprocedures, and used, for example, to treat a patient indicated fortherapy with the antibody.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. There are numerous examples of chemotherapeuticagents that could be conjugated with the therapeutic antibodies of thepresent invention. Examples of such chemotherapeutic agents includealkylating agents such a thiotepa and cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridinessuch as benzodopa, carboquone, meturedopa, and uredopa; ethyleniminesand methylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethylenethiophosphoramide, andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); camptothecin (including the synthetic analoguetopotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,carzelesin, and bizelesin synthetic analogues); cryptophycines(particularly cryptophycin 1 and cryptophycin 8); dolastatin,auristatins, (including analogues monomethyl-auristatin E andmonomethyl-auristatin F); duocarmycin (including the syntheticanalogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin;sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil,chlomaphazine, cholophosphamide, estramustine, ifosfamide,mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,novembichin, phenesterine, prednimustine; trofosfamide, uracil mustard;nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, ranimustine; antibiotics such as the enediyne antibiotics(e.g., calicheamicin, especially calichemicin gamma1I and calicheamicinphi1I, see for example, Agnew, Chem. Intl. Ed. Engl., 33:183-186;dynemicin, including dynemicin A; bisphosphonates, such as clodronate;esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromomophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(Adriamycin™) (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, anddeoxydoxorubicin), epirubucin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycine, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such a methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adranals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; democolcine;diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids suchas maytansine and ansamitocins; mitoguazone, mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitabronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.,paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine (Gemzar™); 6-thioguanine; mercaptopurine;methotrexate; platinum analogs such as cisplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine Navelbine™); novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoids such as retinoic acid; capecitabine; and pharmaceuticallyacceptable salts, acids, or derivatives of any of the above. Alsoincluded in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including Nolvadex™) raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (Fareston™); aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrolacetate (Megace™), exemestane, formestane, fadrozole, vorozole(Rivisor™), letrozole (Femara™), and anastrozole (Arimidex™); andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; and pharmaceutically acceptable salts, acids, orderivatives of any of the above. Any one or more of these agents may beconjugated to the humanized antibodies of the present invention toprovide a useful therapeutic agent for the treatment of variousdisorders.

The antibodies also may be conjugated to prodrugs. A “prodrug” is aprecursor or derivative form of a pharmaceutically active substance thatis less cytotoxic to tumor cells compared to the parent drug and iscapable of being enzymatically activated or converted into the moreactive form. See, for example, Wilman, 1986, “Prodrugs in CancerChemotherapy”, In Biochemical Society Transactions, 14, pp. 375-382,615th Meeting Belfast and Stella et al., 1985, “Prodrugs: A ChemicalApproach to Targeted Drug Delivery, In: “Directed Drug Delivery,Borchardt et al., (ed.), pp. 247-267, Humana Press. Useful prodrugsinclude, but are not limited to, phosphate-containing prodrugs,thiophosphate-containing prodrugs, sulfate-containing prodrugspeptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, β-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs, and optionallysubstituted phenylacetamide-containing prodrugs, 5-fluorocytosine andother 5-fluorouridine prodrugs that can be converted into the moreactive cytotoxic free drug. Examples of cytotoxic drugs that can bederivatized into a prodrug form include, but are not limited to, thosechemotherapeutic agents described above.

For diagnostic as well as therapeutic monitoring purposes, theantibodies of the invention also may be conjugated to a label, either alabel alone or a label and an additional second agent (prodrug,chemotherapeutic agent and the like). A label, as distinguished from theother second agents refers to an agent that is a detectable compound orcomposition and it may be conjugated directly or indirectly to ahumanized antibody of the present invention. The label may itself bedetectable (e.g., radioisotope labels or fluorescent labels) or, in thecase of an enzymatic label, may catalyze chemical alteration of asubstrate compound or composition that is detectable. Labeled humanizedanti-IL-36R antibody can be prepared and used in various applicationsincluding in vitro and in vivo diagnostics.

The antibodies of the present invention may be formulated as part of aliposomal preparation in order to affect delivery thereof in vivo. A“liposome” is a small vesicle composed of various types of lipids,phospholipids, and/or surfactant. Liposomes are useful for delivery to amammal of a compound or formulation, such as a humanized anti-IL-36Rantibody disclosed herein, optionally, coupled to or in combination withone or more pharmaceutically active agents and/or labels. The componentsof the liposome are commonly arranged in a bilayer formation, similar tothe lipid arrangement of biological membranes.

Certain aspects of the present invention related to isolated nucleicacids that encode one or more domains of the humanized antibodies of thepresent invention. An “isolated” nucleic acid molecule is a nucleic acidmolecule that is identified and separated from at least one contaminantnucleic acid molecule with which it is ordinarily associated in thenatural source of the antibody nucleic acid. An isolated nucleic acidmolecule is distinguished from the nucleic acid molecule as it exists innatural cells.

In various aspects of the present invention one or more domains of thehumanized antibodies will be recombinantly expressed. Such recombinantexpression may employ one or more control sequences, i.e.,polynucleotide sequences necessary for expression of an operably linkedcoding sequence in a particular host organism. The control sequencessuitable for use in prokaryotic cells include, for example, promoter,operator, and ribosome binding site sequences. Eukaryotic controlsequences include, but are not limited to, promoters, polyadenylationsignals, and enhancers. These control sequences can be utilized forexpression and production of humanized anti-IL-36R antibody inprokaryotic and eukaryotic host cells.

A nucleic acid sequence is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,a nucleic acid presequence or secretory leader is operably linked to anucleic acid encoding a polypeptide if it is expressed as a preproteinthat participates in the secretion of the polypeptide; a promoter orenhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, “operably linked” means that the DNA sequencesbeing linked are contiguous, and, in the case of a secretory leader,contiguous and in reading frame. However, enhancers are optionallycontiguous. Linking can be accomplished by ligation at convenientrestriction sites. If such sites do not exist, synthetic oligonucleotideadaptors or linkers can be used.

As used herein, the expressions “cell”, “cell line”, and “cell culture”are used interchangeably and all such designations include the progenythereof. Thus, “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers.

The term “mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domesticated and farm animals,and zoo, sports, or pet animals, such as dogs, horses, cats, cows, andthe like. Preferably, the mammal is human.

A “disorder”, as used herein, is any condition that would benefit fromtreatment with a humanized anti-IL-36R antibody described herein. Thisincludes chronic and acute disorders or diseases including thosepathological conditions that predispose the mammal to the disorder inquestion. Non-limiting examples or disorders to be treated hereininclude inflammatory, angiogenic, autoimmune and immunologic disorders,respiratory disorders, cancer, hematological malignancies, benign andmalignant tumors, leukemias and lymphoid malignancies.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.

An IL-36R-associated disorder includes diseases and disorders of theimmune system, such as autoimmune disorders and inflammatory disorders.Such conditions include, but are not limited to, rheumatoid arthritis(RA), systemic lupus erythematosus (SLE), scleroderma, Sjogren'ssyndrome, multiple sclerosis, psoriasis, psoriatic arthritis,inflammatory bowel disease (e.g., ulcerative colitis and Crohn'sdisease), pulmonary inflammation, asthma, idiopathic thrombocytopenicpurara (ITP) epithelial inflammatory disorders, fibrosis and ankylosingspondylitis.

The term “intravenous infusion” refers to introduction of an agent intothe vein of an animal or human patient over a period of time greaterthan approximately 15 minutes, generally between approximately 30 to 90minutes.

The term “intravenous bolus” or “intravenous push” refers to drugadministration into a vein of an animal or human such that the bodyreceives the drug in approximately 15 minutes or less, generally 5minutes or less.

The term “subcutaneous administration” refers to introduction of anagent under the skin of an animal or human patient, preferable within apocket between the skin and underlying tissue, by relatively slow,sustained delivery from a drug receptacle. Pinching or drawing the skinup and away from underlying tissue may create the pocket.

The term “subcutaneous infusion” refers to introduction of a drug underthe skin of an animal or human patient, preferably within a pocketbetween the skin and underlying tissue, by relatively slow, sustaineddelivery from a drug receptacle for a period of time including, but notlimited to, 30 minutes or less, or 90 minutes or less. Optionally, theinfusion may be made by subcutaneous implantation of a drug deliverypump implanted under the skin of the animal or human patient, whereinthe pump delivers a predetermined amount of drug for a predeterminedperiod of time, such as 30 minutes, 90 minutes, or a time periodspanning the length of the treatment regimen.

The term “subcutaneous bolus” refers to drug administration beneath theskin of an animal or human patient, where bolus drug delivery is lessthan approximately 15 minutes; in another aspect, less than 5 minutes,and in still another aspect, less than 60 seconds. In yet even anotheraspect, administration is within a pocket between the skin andunderlying tissue, where the pocket may be created by pinching ordrawing the skin up and away from underlying tissue.

The term “therapeutically effective amount” is used to refer to anamount of an active agent that relieves or ameliorates one or more ofthe symptoms of the disorder being treated. In another aspect, thetherapeutically effective amount refers to a target serum concentrationthat has been shown to be effective in, for example, slowing diseaseprogression. Efficacy can be measured in conventional ways, depending onthe condition to be treated.

The terms “treatment” and “therapy” and the like, as used herein, aremeant to include therapeutic as well as prophylactic, or suppressivemeasures for a disease or disorder leading to any clinically desirableor beneficial effect, including but not limited to alleviation or reliefof one or more symptoms, regression, slowing or cessation of progressionof the disease or disorder. Thus, for example, the term treatmentincludes the administration of an agent prior to or following the onsetof a symptom of a disease or disorder thereby preventing or removing oneor more signs of the disease or disorder. As another example, the termincludes the administration of an agent after clinical manifestation ofthe disease to combat the symptoms of the disease. Further,administration of an agent after onset and after clinical symptoms havedeveloped where administration affects clinical parameters of thedisease or disorder, such as the degree of tissue injury or the amountor extent of metastasis, whether or not the treatment leads toamelioration of the disease, comprises “treatment” or “therapy” as usedherein. Moreover, as long as the compositions of the invention eitheralone or in combination with another therapeutic agent alleviate orameliorate at least one symptom of a disorder being treated as comparedto that symptom in the absence of use of the humanized anti-IL-36Rantibody composition, the result should be considered an effectivetreatment of the underlying disorder regardless of whether all thesymptoms of the disorder are alleviated or not.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

Antibodies

In one aspect, described and disclosed herein are anti-IL-36Rantibodies, in particular humanized anti-IL-36R antibodies, andcompositions and articles of manufacture comprising one or moreanti-IL-36R antibody, in particular one or more humanized anti-IL-36Rantibody of the present invention. Also described are binding agentsthat include an antigen-binding fragment of an anti-IL-36 antibody, inparticular a humanized anti-IL-36R antibody.

Variable regions and CDRs of representative antibodies of the presentinvention are disclosed below:

Anti-IL-36R Mouse Antibody Sequences

Variable regions and CDRs of representative mouse lead antibodies of thepresent invention (mouse leads) are shown below:

Light Chain Variable Region (VK) Amino Acid Sequences >33D10B12vK Protein (antibody 33D10)(SEQ ID NO: 1)QIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQKKPGSSPKLWVYSTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCHQHHRSPVTFGSGTKLEMK >172C8B12 vK protein (antibody 172C8)(SEQ ID NO: 2)DIQMTQSPASQSASLGESVTFTCLASQTIGTWLAWYQQRPGKSPQLLIYAATSLADGVPSRFSGSGSGTQFSFNIRSLQAEDFASYYCQQVYTTPLTFGGGTKLEIK >67E7E8 vK protein (antibody 67E7)(SEQ ID NO: 3)DIQMTQSPASQSASLGESVTFTCLASQTIGTWLGWYQQKPGKSPQLLIYRSTTLADGVPSRFSGSGSGTKFSFKISSLQAADFASYYCQQLYSAPYTFGGGTKLEIR >78C8D1 vK Protein (antibody 78C8)(SEQ ID NO: 4)DVLLTQTPLSLPVSLGDQASISCRSSQNIVHSNGNTYLQWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGAGTKLELK >81A1D1 vK Protein (antibody 81A1)(SEQ ID NO: 5)DIQMTQTTSSLSASLGDRVTISCRASQDIYKYLNWYQQKPDGTLKLLIYYTSGLHSGVPSRFSGSGSGTDFSLTISNLEPEDIATYFCQQDSKFPWTFGGDTKLEIK >81B4E11 vK Protein (antibody 81B4)(SEQ ID NO: 6)QIVLTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLWIYRTSNLASGVPGRFSGSGSGTSYSLTISSMEAEDAATYYCHQFHRSPLTFGAGTKLELK >73C5C10 vK protein (antibody 73C5)(SEQ ID NO: 7)DIVMTQSQKFLSTSVGVRVSVTCKASQDVGTNVLWYQQKIGQSPKPLIYSASYRHSGVPDRFTGSGSGTDFTLIISNVQSEDLAEYFCQQYSRYPLTFGPGTKLELK >73F6F8 vK protein (antibody 73F6)(SEQ ID NO: 8)DIVMTQSQKFLSTSVGVRVSVTCKASQDVGTNVLWYQQKIGQSPKALIYSASYRHSGVPDRFTGSGSGTDFTLIITNVQSEDLAEYFCQQYSRYPLTFGPGTKLELK >76E10E8 vK protein (antibody 76E10)(SEQ ID NO: 9) DIVMTQSQKFMSATVGGRVNITCKASQNVGRAVAWYQQKPGQSPKLLTHSASNRYTGVPDRFTGSGSGTDFTLTITNMQSEDLADYFCQQYSSYPLTFGAGTKLDLK >89A12B8 vK protein (antibody 89A12)(SEQ ID NO: 10)DIQMTQSPASQSASLGESVTFSCLASQTIGTWLGWYQQKPGKSPQLLIYRATSLADGVPSRFSGSGSGTNFSFKISSLQAEDLASYYCQQLYSGPYTFGGGTKLEIRHeavy Chain Variable Region (VH) Amino Acid Sequences >33D10B12vH Protein (antibody 33D10)(SEQ ID NO: 11)QVQLQQSGTELLKPGASVKLSCKASGNTVTSYWMHWVKQRPGQGLEWIGEILPSTGRTNYNENFKGKAMLTVDKSSSTAYMQLSSLASEDSAVYYCTIVYFGNPWHAYWGQGTLVTVSA >172C8B12 vH prolan (antibody 172C8) (SEQ ID NO: 12)EVQLOQSGPELVKPGASVKLSCKASGYTFTDNYMNWVRQSHGKSLEWIGRVNPSNGDTKYNQNFKGKATLTVDKSLSTAYMQLNGLTSEDSAVYYCGRTKNFYSSYSYDDAMDYWGQGTSVTVSS >67E7E8 vH protein (antibody 67E7) (SEQ ID NO: 13)EVQLQQSGAEFVRPGASVKFSCTASGFNIKDDYIHWVRQRPEQGLEWVGRIDPANGNTKYAPKFQDKATITADTSSNTAYLQLSSLTSEDTAVYYCAKSFPNNYYSYDDAFAYWGQGTLVTVSA >78C8D1 vH Protein (antibody 78C8) (SEQ ID NO: 14)QVQLKESGPVLVAPSQSLSITCTVSGFSLTKFGVHWIRQTPGKGLEWLGVIWAGGPTNYNSALMSRLTISKDISQSQVFLRIDSLQTDDTAMYYCAKQIYYSTLVDYWGQGTSVTVSS >81A1D1 vH Protein (antibody 81A1)(SEQ ID NO: 15)QVQLKESGPGLVAPSQSLFITCTVSGFSLSSYEINWVRQVPGKGLEWLGVIWTGITTNYNSALISRLSISKDNSKSLVFLKMNSLQTDDTAIYYCARGTGTGFYYAMDYWGQGTSVTVSS >81B4E11 vH Protein (antibody 81B4) (SEQ ID NO: 16)QVQLQQPGADFVRPGASMRLSCKASGYSFTSSWIHWVKQRPGQGLEWIGEINPGNVRTNYNENFRNKATLTVDKSSTTAYMQLRSLTSADSAVYYCTVVFYGEPYFPYWGQGTLVTVSA >73C5C10 vH Protein (antibody 73C5) (SEQ ID NO: 17)QVQLKESGPGLVAPSQSLSITCTVSGFSLTNYAVHWVRQFPGKGLEWLGVIWSDGSTDFNAPFKSRLSINKDNSKSQVFFKMNSLQIDDTAIYYCARKGGYSGSWFAYWGQGTLVTVSA >73F6F8 vH protein (antibody 73F6) (SEQ ID NO: 18)QVQLKESGPGLVAPSQSLSITCTVSGFSLTNYAVHWVRQFPGKGLEWLGVIWSDGSTDYNAPFKSRLSINKDNSKSQVFFKMNSLQTDDTAIYYCARKGGYSGSWFAYWGQGTLVTVSA >76E10E8 vH protein (antibody 76E10) (SEQ ID NO: 19)VQLKESGPVLVAPSQSLSITCTVSGFSLTNYGVHWVRQPPGKGLEWLGVIWPVGSTNYNSALMSRLSIHKDNSKSQVFLRMNSLQTDDTAIYYCAKMDWDDFFDYWGQGTTLTVSS >89A12B8 vH Protein (antibody 89A12)(SEQ ID NO: 20)EVQLQQSGAELVRPGASVRLSCTASGFNIKDDYIHWVRQRPKQGLEWLGRIDPANGNTKYDPRFQDKATITADTSSNTAYLHLSSLTSEDTAVYYCAKSFPDNYYSYDDAFAYWGQ GTLVTVSALight chain CDR-1 (L-CDR1) Amino Acid Sequences >33D10G1 L-CDR1(SEQ ID NO: 21) TASSSVSSSYLH >172C8B12 L-CDR1 (SEQ ID NO: 22)LASQTIGTWLA >67E7E8 L-CDR1 (SEQ ID NO: 23) LASQTIGTWLG >78C8D1 L-CDR1(SEQ ID NO: 24) RSSQNIVHSNGNTYLQ >81A1D1 L-CDR1 (SEQ ID NO: 25)RASQDIYKYLN >81B4E11 L-CDR1 (SEQ ID NO: 26) TASSSVSSSYFH >73C5C10 L-CDR1(SEQ ID NO: 27) KASQDVGTNVL >73F6F8 L-CDR1 (SEQ ID NO: 27)KASQDVGTNVL >76E10E8 L-CDR1 (SEQ ID NO: 28) KASQNVGRAVA >89A12B8 L-CDR1(SEQ ID NO: 29) LASQTIGTWLGLight chain CDR-2 (L-CDR2) Amino Acid Sequences >33D10B12 L-CDR2(SEQ ID NO: 30) STSNLAS >172C8B12 L-CDR2 (SEQ ID NO: 31)AATSLAD >67E7E8 L-CDR2 (SEQ ID NO: 32) RSTTLAD >78C8D1 L-CDR2(SEQ ID NO: 33) KVSNRFS >81A1D1 L-CDR2 (SEQ ID NO: 34)YTSGLHS >81B4E11 L-CDR2 (SEQ ID NO: 35) RTSNLAS >73C5C10 L-CDR2(SEQ ID NO: 36) SASYRHS >73F6F8 L-CDR2 (SEQ ID NO: 36)SASYRHS >76E10E8 L-CDR2 (SEQ ID NO: 37) SASNRYT >89A12B8 L-CDR2(SEQ ID NO: 38) RATSLADLight chain CDR-3 (L-CDR3) Amino Acid Sequences >33D10B12 L-CDR3(SEQ ID NO: 39) HQHHRSPVT >172C8B12 L-CDR3 (SEQ ID NO: 40)QQVYTTPLT >67E7E8 L-CDR3 (SEQ ID NO: 41) QQLYSAPYT >78C8D1 L-CDR3(SEQ ID NO: 42) FQGSHVPFT >81A1D1 L-CDR3 (SEQ ID NO: 43)QQDSKFPWT >81B4E11 L-CDR3 (SEQ ID NO: 44) HQFHRSPLT >73C5C10 L-CDR3(SEQ ID NO: 45) QQYSRYPLT >73F6F8 L-CDR3 (SEQ ID NO: 45)QQYSRYPLT >76E10E8 L-CDR3 (SEQ ID NO: 46) QQYSSYPLT >89A12B8 L-CDR3(SEQ ID NO: 47) QQLYSGPYTHeavy chain CDR-1 (H-CDR1) Amino Acid Sequences >33D10B12 H-CDR1(SEQ ID NO: 48) GNTVTSYWMH >172C8B12 H-CDR1 (SEQ ID NO: 49)GYTFTDNYMN >67E7E8 H-CDR1 (SEQ ID NO: 50) GFNIKDDYIH >78C8D1 H-CDR1(SEQ ID NO: 51) GFSLTKFGVH >81A1D1 H-CDR1 (SEQ ID NO: 52)GFSLSSYEIN >81B4E11 H-CDR1 (SEQ ID NO: 53) GYSFTSSWIH >73C5C10 H-CDR1(SEQ ID NO: 54) GFSLTNYAVH >73F6F8 H-CDR1 (SEQ ID NO: 54)GFSLTNYAVH >76E10E8 H-CDR1 (SEQ ID NO: 55) GFSLTNYGVH >89A12B8 H-CDR1(SEQ ID NO: 56) GFNIKDDYIHHeavy chain CDR-2 (H-CDR2) Amino Acid Sequences >33D10B12 H-CDR2(SEQ ID NO: 57) EILPSTGRTNYNENFKG >172C8B12 H-CDR2 (SEQ ID NO: 58)RVNPSNGDTKYNQNFKG >67E7E8 H-CDR2 (SEQ ID NO: 59)RIDPANGNTKYAPKFQD >78C8D1 H-CDR2 (SEQ ID NO: 60)VIWAGGPTNYNSALMS >81A1D1 H-CDR2 (SEQ ID NO: 61)VIWTGITTNYNSALIS >81B4E11 H-CDR2 (SEQ ID NO: 62)EINPGNVRTNYNENF >73C5C10 H-CDR2 (SEQ ID NO: 63)VIWSDGSTDFNAPFKS >73F6F8 H-CDR2 (SEQ ID NO: 64)VIWSDGSTDYNAPFKS >76E10E8 H-CDR2 (SEQ ID NO: 65)VIWPVGSTNYNSALMS >89A12B8 H-CDR2 (SEQ ID NO: 66) RIDPANGNTKYDPRFQDHeavy chain CDR-3 (H-CDR3) Amino Acid Sequences >33D10B12 H-CDR3(SEQ ID NO: 67) VYFGNPWFAY >172C8B12 H-CDR3 (SEQ ID NO: 68)TKNFYSSYSYDDAMDY >67E7E8 H-CDR3 (SEQ ID NO: 69)SFPNNYYSYDDAFAY >78C8D1 H-CDR3 (SEQ ID NO: 70) QIYYSTLVDY >81A1D1 H-CDR3(SEQ ID NO: 71) GTGTGFYYAMDY >81B4E11 H-CDR3 (SEQ ID NO: 72)VFYGEPYFPY >73C5C10 H-CDR3 (SEQ ID NO: 73) KGGYSGSWFAY >73F6F8 H-CDR3(SEQ ID NO: 73) KGGYSGSWFAY >76E10E8 H-CDR3 (SEQ ID NO: 74)MDWDDFFDY >89A12B8 H-CDR3 (SEQ ID NO: 75) SFPDNYYSYDDAFAY

Anti-IL-36R Mouse CDR Sequences A Summary of the CDR Sequences of theLead Mouse Antibodies is Shown Below:

Antibody H-CDR Sequences L-CDR Sequences 33D10 GNTVTSYWMH (H-CDR1)TASSSVSSSYLH (L-CDR1) SEQ ID No: 48 SEQ ID No: 21 EILPSTGRTNYNENFKGSTSNLAS (L-CDR2) SEQ ID (H-CDR2) SEQ ID No: 57 No: 30VYFGNPWFAY (H-CDR3) HQHHRSPVT (L-CDR3) SEQ ID No: 67 SEQ ID No: 39 172C8GYTFTDNYMN (H-CDR1) LASQTIGTWLA (L-CDR1) SEQ ID No: 49 SEQ ID No: 22RVNPSNGDTKYNQNFKG AATSLAD (L-CDR2) SEQ (H-CDR2) SEQ ID No: 58 ID No: 31TKNFYSSYSYDDAMDY QQVYTTPLT (L-CDR3) (H-CDR3) SEQ ID No: 68 SEQ ID No: 4067E7 GFNIKDDYIH (H-CDR1) LASQTIGTWLG (L-CDR1) SEQ ID No: 50SEQ ID No: 23 RIDPANGNTKYAPKFQD RSTTLAD (L-CDR2) SEQ ID(H-CDR2) SEQ ID No: 59 No: 32 SFPNNYYSYDDAFAY (H- QQLYSAPYT (L-CDR3)CDR3) SEQ ID No: 69 SEQ ID No: 41 78C8 GFSLTKFGVH (H-CDR1)RSSQNIVHSNGNTYLQ (L- SEQ ID No: 51 CDR1) SEQ ID No: 24 VIWAGGPTNYNSALMSKVSNRFS (L-CDR2) SEQ ID (H-CDR2) SEQ ID No: 60 No: 33QIYYSTLVDY (H-CDR3) FQGSHVPFT (L-CDR3) SEQ SEQ ID No: 70 ID No: 42 81A1GFSLSSYEIN (H-CDR1) RASQDIYKYLN (L-CDR1) SEQ ID No: 52 SEQ ID No: 25VIWTGITTNYNSALIS (H- YTSGLHS (L-CDR2) SEQ ID CDR2) SEQ ID No: 61 No: 34GTGTGFYYAMDY (H- QQDSKFPWT (L-CDR3) CDR3) SEQ ID No: 71 SEQ ID No: 4381B4 GYSFTSSWIH (H-CDR1) TASSSVSSSYFH (L-CDR1) SEQ ID No: 53SEQ ID No: 26 EINPGNVRTNYNENF (H- RTSNLAS (L-CDR2) SEQ IDCDR2) SEQ ID No: 62 No: 35 VFYGEPYFPY (H-CDR3) HQFHRSPLT (L-CDR3) SEQSEQ ID No: 72 ID No: 44 73C5 GFSLTNYAVH (H-CDR1) KASQDVGTNVL (L-CDR1)SEQ ID No: 54 SEQ ID No: 27 VIWSDGSTDFNAPFKS (H- SASYRHS (L-CDR2) SEQ IDCDR2) SEQ ID No: 63 No: 36 KGGYSGSWFAY (H-CDR3) QQYSRYPLT (L-CDR3)SEQ ID No: 73 SEQ ID No: 45 73F6 GFSLTNYAVH (H-CDR1)KASQDVGTNVL (L-CDR1) SEQ ID No: 54 SEQ ID No: 27 VIWSDGSTDYNAPFKS (H-SASYRHS (L-CDR2) SEQ ID CDR2) SEQ ID No: 64 No: 36 KGGYSGSWFAY (H-CDR3)QQYSRYPLT (L-CDR3) SEQ ID No: 73 SEQ ID No: 45 76E10 GFSLTNYGVH (H-CDR1)KASQNVGRAVA (L-CDR1) SEQ ID No: 55 SEQ ID No: 28 VIWPVGSTNYNSALMS (H-SASNRYT (L-CDR2) SEQ CDR2) SEQ ID No: 65 ID No: 37 MDWDDFFDY (H-CDR3)QQYSSYPLT (L-CDR3) SEQ SEQ ID No: 74 ID No: 46 89A12 GFNIKDDYIH (H-CDR1)LASQTIGTWLG (L-CDR1) SEQ ID No: 56 SEQ ID No: 29 RIDPANGNTKYDPRFQDRATSLAD (L-CDR2) SEQ (H-CDR2) SEQ ID No: 66 ID No: 38SFPDNYYSYDDAFAY (H- QQLYSGPYT (L-CDR3) CDR3) SEQ ID No: 75 SEQ ID No: 47

Anti-IL-36R Humanized Antibody Sequences

Human framework sequences were selected for the mouse leads based on theframework homology, CDR structure, conserved canonical residues,conserved interface packing residues and other parameters to producehumanized variable regions (see Example 5).

Representative humanized variable regions derived from antibodies 81B4and 73C5 are shown below.

Light Chain Variable Region (VK) Amino Acid Sequences >81B4vK32_3 vK protein(SEQ ID NO: 76)EIVLTQSPGTLSLSPGERATMSCTASSSVSSSYFHWYQQKPGQAPRLLIYRTSTLASGIPDRFSGSGSGTDFTLTISRLEPEDAATYYCHQFHRSPLTFGQGTKLEIK >81B4vK32_105 vK protein(SEQ ID NO: 77)EIVLTQSPGTLSLSPGERATMSCTASSSVSSSYFHWYQQKPGQAPRLLIYRTSILASGVPDRFSGSGSGTDFTLTISRLEPEDFATYYCHQFHRSPLTFGQGTKLEIK >81B4vK32_116 vK protein(SEQ ID NO: 78)EIVLTQSPGTLSLSPGERATMSCTASSSVSSSYFHWYQQKPGQAPRLWIYRTSRLASGVPDRFSGSGSGTDFTLTISRLEPEDAATYYCHQFHRSPLTFGQGTKLEIK >81B4vK32_127 vK protein(SEQ ID NO: 79)EIVLTQSPGTLSLSPGERATMTCTASSSVSSSYFHWYQQKPGQAPRLLIYRTSRLASGVPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQFHRSPLTFGQGTKLEIK >81B4vK32_138 vK protein(SEQ ID NO: 80)QIVLTQSPGTLSLSPGERATMTCTASSSVSSSYFHWYQQKPGQAPRLWIYRTSRLASGVPDRFSGSGSGTDFTLTISRLEPEDAATYYCHQFHRSPLTFGAGTKLEIK >81B4vK32_140 vK protein(SEQ ID NO: 81)QIVLTQSPGTLSLSPGERVTMSCTASSSVSSSYFHWYQQKPGQAPRLLIYRTSQLASGIPDRFSGSGSGTDFTLTISRLEPEDAATYYCHQFHRSPLTFGQGTKLEIK >81B4vK32_141 vK protein(SEQ ID NO: 82)QIVLTQSPGTLSLSPGERATMTCTASSSVSSSYFHWYQQKPGQAPRLLIYRTSKLASGVPDRFSGSGSGTDFTLTISRLEPEDFATYYCHQFHRSPLTFGQGTKLEIK >81B4vK32_147 vK protein(SEQ ID NO: 83)EIVLTQSPGTLSLSPGERATMSCTASSSVSSSYFHWYQQKPGQAPRLLIYRTSHLASGIPGRFSGSGSGTDFTLTISRLEPEDAAVYYCHQFHRSPLTFGQGTKLEIK >73C5vK39_2 vK protein(SEQ ID NO: 84)EIVMTQSPATLSVSPGVRATLSCKASQDVGTNVLWYQQKPGQAPRPLIYSASYRHSGIPDRFSGSGSGTEFTLTISSLQSEDFAEYFCQQYSRYPLTFGQGTKLEIK >73C5vK39_7 vK protein(SEQ ID NO: 85)EIVMTQSPATLSVSPGVRATLSCKASQDVGTNVLWYQQKPGQAPRPLIYSASYRHSGIPDRFSGSGSGTEFTLTISSLQSEDFAVYYCQQYSRYPLTFGQGTKLEIK >73C5vK39_15 vK protein(SEQ ID NO: 86)EIVMTQSPATLSVSPGVRATLSCKASQDVGTNVLWYQQKPGQAPRPLIYSASYRHSGIPARFSGSGSGTEFTLTISSLQSEDFAEYYCQQYSRYPLTFGQGTKLEIKHeavy Chain Variable Region (VH) Amino Acid Sequences >81B4vH33_49 vH Protein(SEQ ID NO: 87)QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVRQAPGQGLEWIGEINPGNVRTNYNENFRNKATMTVDTSISTAYMELSRLRSDDTAVYYCAVVFYGEPYFPYWGQGTLVTVSS  >81B4vH33_85T vH Protein (SEQ ID NO: 88)QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVRQRPGQGLEWIGEINPGNVRTNYNENFRNRVTMTVDTSISTAYMELSRLRSDDTAVYYCTVVFYGEPYFPYWGQGTLVTVSS  >81B4vH33_90 vH Protein (SEQ ID NO: 89)QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVKQAPGQGLEWMGEINPGNVRTNYNENFRNKVTMTVDTSISTAYMELSRLRSDDTAVYYCTVVFYGEPYFPYWGQGTLVTVSS  >81B4vH33_93 vH Protein (SEQ ID NO: 90)QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVRQRPGQGLEWMGEINPGNVRTNYNENFRNRATLTRDTSISTAYMELSRLRSDDTAVYYCAVVFYGEPYFPYWGQGTLVTVSS  >81B4vH50_22 vH Protein (SEQ ID NO: 91)QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVRQRPGQGLEWMGEILPGVVRTNYNENFRNKVTMTVDTSISTAYMELSRLRSDDTAVYYCTVVFYGEPYFPYWGQGTLVTVSS  >81B4vH50_30 vH Protein (SEQ ID NO: 92)QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVRQRPGQGLEWIGEINPGAVRTNYNENFRNRVTMTVDTSISTAYMELSRLRSDDTAVYYCTVVFYGEPYFPYWGQGTLVTVSS  >81B4vH51_13 vH Protein (SEQ ID NO: 93)QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVRQAPGQGLEWIGEINPGLVRTNYNENFRNKVTMTVDTSISTAYMELSRLRSDDTAVYYCAVVFYGEPYFPYWGQGTLVTVSS  >81B4vH51_15 vH Protein (SEQ ID NO: 94)QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVRQAPGQGLEWIGEINPGAVRTNYNENFRNKVTMTVDTSISTAYMELSRLRSDDTAVYYCAVVFYGEPYFPYWGQGTLVTVSS  >81B4vH52_83 vH Protein (SEQ ID NO: 95)QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVRQAPGQGLEWIGEINPGSVRTNYNENFRNKATMTVDTSISTAYMELSRLRSDDTAVYYCAVVFYGEPYFPYWGQGTLVTVSS  >73C5vH46_4 vH Protein (SEQ ID NO: 96)QVQLQESGPGLVKPSETLSITCTVSGFSLTDYAVHWIRQPPGKGLEWIGVIWSDGSTDYNAPFKSRVTINKDTSKSQVSFKMSSVQAADTAVYYCARKGGYSGSWFAYWGQGTLVTVSS  >73C5vH46_19 vH Protein (SEQ ID NO: 97)QVQLQESGPGLVKPSETLSITCTVSGFSLTDYAVHWIRQPPGKGLEWIGVIWSDGSTDYNAPFKSRVTISKDTSKNQVSLKMNSLTTDDTAVYYCARKGGYSGSWFAYWGQGTLVTVSS  >73C5vH46_40 vH Protein (SEQ ID NO: 98)QVQLQESGPGLVKPSETLSITCTVSGFSLTDYAVHWIRQPPGKGLEWIGVIWSDGSTDYNAPFKSRVTISKDNSKSQVSLKMNSVTVADTAVYYCARKGGYSGSWFAYWGQGTLVTVSS  >73C5vH47_65 vH Protein (SEQ ID NO: 99)QVQLQESGPGLVKPSETLSITCTVSGFSLTDYAVHWVRQPPGKGLEWIGVIWSDGSTDYNAPFKSRVTISKDTSKNQVSFKLSSVTVDDTAVYYCARKGGYSGSWFAYWGQGTLVTVSS  >73C5vH47_77 vH Protein (SEQ ID NO: 100)QVQLQESGPGLVAPSETLSLTCTVSGFSLTDYAVHWIRQFPGKGLEWIGVIWSDGSTDFNAPFKSRVTISKDTSKNQVSFKLSSVTTDDTAVYYCARKGGYSGSWFAYWGQGTLVTVSS  >73C5vH58_91 vH Protein (SEQ ID NO: 101)QVQLQESGPGLVKPSETLSITCTVSGFSLTDYAVHWIRQPPGKGLEWIGVIWSDGSTDYNAPFKSRVTISKDNSKSQVSFKMSSVTADDTAVYYCARKGGYSGSWFAYWGQGTLVTVS S 

The CDR sequences from the humanized variable regions derived fromantibodies 81B4 and 73C5 shown above are depicted below.

L-CDR1 Amino Acid Sequences

>81B4vK32_3 L-CDR1 TASSSVSSSYFH (SEQ ID NO: 26) >81B4vK32_105 L-CDR1TASSSVSSSYFH (SEQ ID NO: 26) >81B4vK32_116 L-CDR1 TASSSVSSSYFH(SEQ ID NO: 26) >81B4vK32_127 L-CDR1 TASSSVSSSYFH(SEQ ID NO: 26) >81B4vK32_138 L-CDR1 TASSSVSSSYFH(SEQ ID NO: 26) >81B4vK32_140 L-CDR1 TASSSVSSSYFH(SEQ ID NO: 26) >81B4vK32_141 L-CDR1 TASSSVSSSYFH(SEQ ID NO: 26) >81B4vK32_147 L-CDR1 TASSSVSSSYFH(SEQ ID NO: 26) >73C5vK39_2 L-CDR1 KASQDVGTNVL(SEQ ID NO: 27) >73C5vK39_7 L-CDR1 KASQDVGTNVL(SEQ ID NO: 27) >73C5vK39_15 L-CDR1 KASQDVGTNVL (SEQ ID NO: 27)

L-CDR2 Amino Acid Sequences

>81B4vK32_3 L-CDR2 (SEQ ID 102) RTSTLAS >81B4vK32_105 L-CDR2(SEQ ID 103) RTSILAS >81B4vK32_116 L-CDR2 (SEQ ID 104)RTSRLAS >81B4vK32_127 L-CDR2 (SEQ ID 104) RTSRLAS >81B4vK32_138 L-CDR2(SEQ ID 104) RTSRLAS >81B4vK32_140 L-CDR2 (SEQ ID 105)RTSQLAS >81B4vK32_141 L-CDR2 (SEQ ID 106) RTSKLAS >81B4vK32_147 L-CDR2(SEQ ID 140) RTSHLAS >73C5vK39_2 L-CDR2 SASYRHS(SEQ ID NO: 36) >73C5vK39_7 L-CDR2 SASYRHS(SEQ ID NO: 36) >73C5vK39_15 L-CDR2 SASYRHS (SEQ ID NO: 36)

L-CDR3 Amino Acid Sequences

>81B4vK32_3 L-CDR3 HQFHRSPLT (SEQ ID NO: 44) >81B4vK32_105 L-CDR3HQFHRSPLT (SEQ ID NO: 44) >81B4vK32_116 L-CDR3 HQFHRSPLT(SEQ ID NO: 44) >81B4vK32_127 L-CDR3 HQFHRSPLT(SEQ ID NO: 44) >81B4vK32_138 L-CDR3 HQFHRSPLT(SEQ ID NO: 44) >81B4vK32_140 L-CDR3 HQFHRSPLT(SEQ ID NO: 44) >81B4vK32_141 L-CDR3 HQFHRSPLT(SEQ ID NO: 44) >81B4vK32_147 L-CDR3 HQFHRSPLT(SEQ ID NO: 44) >73C5vK39_2 L-CDR3 QQYSRYPLT(SEQ ID NO: 45) >73C5vK39_7 L-CDR3 QQYSRYPLT(SEQ ID NO: 45) >73C5vK39_15 L-CDR3 QQYSRYPLT (SEQ ID NO: 45)

H-CDR1 Amino Acid Sequences

>81B4vH33_49 H-CDR1 GYSFTSSWIH (SEQ ID NO: 53) >81B4vH33_85T H-CDR1GYSFTSSWIH (SEQ ID NO: 53) >81B4vH33_90 H-CDR1 GYSFTSSWIH(SEQ ID NO: 53) >81B4vH33_93 H-CDR1 GYSFTSSWIH(SEQ ID NO: 53) >81B4vH50_22 H-CDR1 GYSFTSSWIH(SEQ ID NO: 53) >81B4vH50_30 H-CDR1 GYSFTSSWIH(SEQ ID NO: 53) >81B4vH51_13 H-CDR1 GYSFTSSWIH(SEQ ID NO: 53) >81B4vH51_15 H-CDR1 GYSFTSSWIH(SEQ ID NO: 53) >81B4vH52_83 H-CDR1 GYSFTSSWIH(SEQ ID NO: 53) >73C5vH46_4 H-CDR1 GFSLTDYAVH(SEQ ID NO: 107) >73C5vH46_19 H-CDR1 GFSLTDYAVH(SEQ ID NO: 107) >73C5vH46_40 H-CDR1 GFSLTDYAVH(SEQ ID NO: 107) >73C5vH47_65 H-CDR1 GFSLTDYAVH(SEQ ID NO: 107) >73C5vH47_77 H-CDR1 GFSLTDYAVH(SEQ ID NO: 107) >73C5vH58_91 H-CDR1 GFSLTDYAVH (SEQ ID NO: 107)

H-CDR2 Amino Acid Sequences

>81B4vH33_49 H-CDR2 EINPGNVRTNYNENF (SEQ ID NO: 62) >81B4vH33_85T H-CDR2EINPGNVRTNYNENF (SEQ ID NO: 62) >81B4vH33_90 H-CDR2 EINPGNVRTNYNENF(SEQ ID NO: 62) >81B4vH33_93 H-CDR2 EINPGNVRTNYNENF(SEQ ID NO: 62) >81B4vH50_22 H-CDR2 EILPGVVRTNYNENF(SEQ ID NO: 108) >81B4vH50_30 H-CDR2 EINPGAVRTNYNENF(SEQ ID NO: 109) >81B4vH51_13 H-CDR2 EINPGLVRTNYNENF(SEQ ID NO: 110) >81B4vH51_15 H-CDR2 EINPGAVRTNYNENF(SEQ ID NO: 109) >81B4vH52_83 H-CDR2 EINPGSVRTNYNENF(SEQ ID NO: 111) >73C5vH46_4 H-CDR2 VIWSDGSTDYNAPFKS(SEQ ID NO: 64) >73C5vH46_19 H-CDR2 VIWSDGSTDYNAPFKS(SEQ ID NO: 64) >73C5vH46_40 H-CDR2 VIWSDGSTDYNAPFKS(SEQ ID NO: 64) >73C5vH47_65 H-CDR2 VIWSDGSTDYNAPFKS(SEQ ID NO: 64) >73C5vH47_77 H-CDR2 VIWSDGSTDFNAPFKS(SEQ ID NO: 63) >73C5vH58_91 H-CDR2 VIWSDGSTDYNAPFKS (SEQ ID NO: 64)

H-CDR3 Amino Acid Sequences

>81B4vH33_49 H-CDR3 VFYGEPYFPY (SEQ ID NO: 72) >81B4vH33_85T H-CDR3VFYGEPYFPY (SEQ ID NO: 72) >81B4vH33_90 H-CDR3 VFYGEPYFPY(SEQ ID NO: 72) >81B4vH33_93 H-CDR3 VFYGEPYFPY(SEQ ID NO: 72) >81B4vH50_22 H-CDR3 VFYGEPYFPY(SEQ ID NO: 72) >81B4vH50_30 H-CDR3 VFYGEPYFPY(SEQ ID NO: 72) >81B4vH51_13 H-CDR3 VFYGEPYFPY(SEQ ID NO: 72) >81B4vH51_15 H-CDR3 VFYGEPYFPY(SEQ ID NO: 72) >81B4vH52_83 H-CDR3 VFYGEPYFPY(SEQ ID NO: 72) >73C5vH46_4 H-CDR3 KGGYSGSWFAY(SEQ ID NO: 73) >73C5vH46_19 H-CDR3 KGGYSGSWFAY(SEQ ID NO: 73) >73C5vH46_40 H-CDR3 KGGYSGSWFAY(SEQ ID NO: 73) >73C5vH47_65 H-CDR3 KGGYSGSWFAY(SEQ ID NO: 73) >73C5vH47_77 H-CDR3 KGGYSGSWFAY(SEQ ID NO: 73) >73C5vH58_91 H-CDR3 KGGYSGSWFAY (SEQ ID NO: 73)

In one aspect, a variable region of the present invention is linked to aconstant region. For example, a variable region of the present inventionis linked to a constant region shown below to form a heavy chain or alight chain of an antibody.

Heavy Chain Constant region linked downstream of a humanized variableheavy region: (SEQ ID NO: 112)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light Chain Constant region linkeddownstream of a humanized variable light region: (SEQ ID NO: 113)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC

Representative light chain and heavy chain sequences of the presentinvention are shown below (humanized variable regions derived fromantibodies 81B4 and 73C5 linked to constant regions).

Light Chain Amino Acid Sequences >81B4vK32_3 Light Chain(SEQ ID NO: 114)EIVLTQSPGTLSLSPGERATMSCTASSSVSSSYFHWYQQKPGQAPRLLIYRTSTLASGIPDRFSGSGSGTDFTLTISRLEPEDAATYYCHQFHRSPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >81B4vK32_105 Light Chain(SEQ ID NO: 115)EIVLTQSPGTLSLSPGERATMSCTASSSVSSSYFHWYQQKPGQAPRLLIYRTSILASGVPDRFSGSGSGTDFTLTISRLEPEDFATYYCHQFHRSPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >81B4vK32_116 Light Chain(SEQ ID NO: 116)EIVLTQSPGTLSLSPGERATMSCTASSSVSSSYFHWYQQKPGQAPRLWIYRTSRLASGVPDRFSGSGSGTDFTLTISRLEPEDAATYYCHQFHRSPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >81B4vK32_127 Light Chain(SEQ ID NO: 117)EIVLTQSPGTLSLSPGERATMTCTASSSVSSSYFHWYQQKPGQAPRLLIYRTSRLASGVPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQFHRSPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >81B4vK32_138 Light Chain(SEQ ID NO: 118)QIVLTQSPGTLSLSPGERATMTCTASSSVSSSYFHWYQQKPGQAPRLWIYRTSRLASGVPDRFSGSGSGTDFTLTISRLEPEDAATYYCHQFHRSPLTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >81B4vK32_140 Light Chain(SEQ ID NO: 119)QIVLTQSPGTLSLSPGERVTMSCTASSSVSSSYFHWYQQKPGQAPRLLIYRTSQLASGIPDRFSGSGSGTDFTLTISRLEPEDAATYYCHQFHRSPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >81B4vK32_141 Light Chain(SEQ ID NO: 120)QIVLTQSPGTLSLSPGERATMTCTASSSVSSSYFHWYQQKPGQAPRLLIYRTSKLASGVPDRFSGSGSGTDFTLTISRLEPEDFATYYCHQFHRSPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >81B4vK32_147 Light Chain(SEQ ID NO: 121)EIVLTQSPGTLSLSPGERATMSCTASSSVSSSYFHWYQQKPGQAPRLLIYRTSHLASGIPGRFSGSGSGTDFTLTISRLEPEDAAVYYCHQFHRSPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >73C5vK39_2 Light Chain(SEQ ID NO: 122)EIVMTQSPATLSVSPGVRATLSCKASQDVGTNVLWYQQKPGQAPRPLIYSASYRHSGIPDRFSGSGSGTEFTLTISSLQSEDFAEYFCQQYSRYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >73C5vK39_7 Light Chain(SEQ ID NO: 123)EIVMTQSPATLSVSPGVRATLSCKASQDVGTNVLWYQQKPGQAPRPLIYSASYRHSGIPDRFSGSGSGTEFTLTISSLQSEDFAVYYCQQYSRYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >73C5vK39_15 Light Chain(SEQ ID NO: 124)EIVMTQSPATLSVSPGVRATLSCKASQDVGTNVLWYQQKPGQAPRPLIYSASYRHSGIPARFSGSGSGTEFTLTISSLQSEDFAEYYCQQYSRYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECHeavy Chain Amino Acid Sequences >81B4vH33_49 Heavy Chain(SEQ ID NO: 125)QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVRQAPGQGLEWIGEINPGNVRTNYNENFRNKATMTVDTSISTAYMELSRLRSDDTAVYYCAVVFYGEPYFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >81B4vH33_85T Heavy Chain(SEQ ID NO: 126)QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVRQRPGQGLEWIGEINPGNVRTNYNENFRNRVTMTVDTSISTAYMELSRLRSDDTAVYYCTVVFYGEPYFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >81B4vH33_90 Heavy Chain(SEQ ID NO: 127)QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVKQAPGQGLEWMGEINPGNVRTNYNENFRNKVTMTVDTSISTAYMELSRLRSDDTAVYYCTVVFYGEPYFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >81B4vH33_93 Heavy Chain(SEQ ID NO: 128)QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVRQRPGQGLEWMGEINPGNVRTNYNENFRNRATLTRDTSISTAYMELSRLRSDDTAVYYCAVVFYGEPYFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >81B4vH50_22 Heavy Chain(SEQ ID NO: 129)QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVRQRPGQGLEWMGEILPGVVRTNYNENFRNKVTMTVDTSISTAYMELSRLRSDDTAVYYCTVVFYGEPYFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >81B4vH50_30 Heavy Chain(SEQ ID NO: 130)QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVRQRPGQGLEWIGEINPGAVRTNYNENFRNRVTMTVDTSISTAYMELSRLRSDDTAVYYCTVVFYGEPYFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >81B4vH51_13 Heavy Chain(SEQ ID NO: 131)QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVRQAPGQGLEWIGEINPGLVRTNYNENFRNKVTMTVDTSISTAYMELSRLRSDDTAVYYCAVVFYGEPYFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >81B4vH51_15 Heavy Chain(SEQ ID NO: 132)QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVRQAPGQGLEWIGEINPGAVRTNYNENFRNKVTMTVDTSISTAYMELSRLRSDDTAVYYCAVVFYGEPYFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >81B4vH52_83 Heavy Chain(SEQ ID NO: 133)QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVRQAPGQGLEWIGEINPGSVRTNYNENFRNKATMTVDTSISTAYMELSRLRSDDTAVYYCAVVFYGEPYFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >73C5vH46_4 Heavy Chain(SEQ ID NO: 134)QVQLQESGPGLVKPSETLSITCTVSGFSLTDYAVHWIRQPPGKGLEWIGVIWSDGSTDYNAPFKSRVTINKDTSKSQVSFKMSSVQAADTAVYYCARKGGYSGSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >73C5vH46_19 Heavy Chain(SEQ ID NO: 135)QVQLQESGPGLVKPSETLSITCTVSGFSLTDYAVHWIRQPPGKGLEWIGVIWSDGSTDYNAPFKSRVTISKDTSKNQVSLKMNSLTTDDTAVYYCARKGGYSGSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >73C5vH46_40 Heavy Chain(SEQ ID NO: 136)QVQLQESGPGLVKPSETLSITCTVSGFSLTDYAVHWIRQPPGKGLEWIGVIWSDGSTDYNAPFKSRVTISKDNSKSQVSLKMNSVTVADTAVYYCARKGGYSGSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >73C5vH47_65 Heavy Chain(SEQ ID NO: 137)QVQLQESGPGLVKPSETLSITCTVSGFSLTDYAVHWVRQPPGKGLEWIGVIWSDGSTDYNAPFKSRVTISKDTSKNQVSFKLSSVTVDDTAVYYCARKGGYSGSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >73C5vH47_77 Heavy Chain(SEQ ID NO: 138)QVQLQESGPGLVAPSETLSLTCTVSGFSLTDYAVHWIRQFPGKGLEWIGVIWSDGSTDFNAPFKSRVTISKDTSKNQVSFKLSSVTTDDTAVYYCARKGGYSGSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >73C5vH58_91 Heavy Chain(SEQ ID NO: 139)QVQLQESGPGLVKPSETLSITCTVSGFSLTDYAVHWIRQPPGKGLEWIGVIWSDGSTDYNAPFKSRVTISKDNSKSQVSFKMSSVTADDTAVYYCARKGGYSGSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKThe CDRs listed above are defined using the Chothia numbering system (Al-Lazikani et al., (1997)  JMB 273, 927-948).

In one aspect, an antibody of the present invention comprises 3 lightchain CDRs and 3 heavy chain CDRs, for example as set forth above.

In one aspect, an antibody of the present invention comprises a lightchain and a heavy chain variable region as set forth above. In oneaspect, a light chain variable region of the invention is fused to alight chain constant region, for example a kappa or lambda constantregion. In one aspect, a heavy chain variable region of the invention isfused to a heavy chain constant region, for example IgA, IgD, IgE, IgGor IgM, in particular, IgG₁, IgG₂, IgG₃ or IgG₄.

The present invention provides an anti-IL-36R antibody comprising alight chain comprising the amino acid sequence of SEQ ID NO: 115; and aheavy chain comprising the amino acid sequence of SEQ ID NO: 125(Antibody B1).

The present invention provides an anti-IL-36R antibody comprising alight chain comprising the amino acid sequence of SEQ ID NO: 115; and aheavy chain comprising the amino acid sequence of SEQ ID NO: 126(Antibody B2).

The present invention provides an anti-IL-36R antibody comprising alight chain comprising the amino acid sequence of SEQ ID NO: 115; and aheavy chain comprising the amino acid sequence of SEQ ID NO: 127(Antibody B3).

The present invention provides an anti-IL-36R antibody comprising alight chain comprising the amino acid sequence of SEQ ID NO: 118; and aheavy chain comprising the amino acid sequence of SEQ ID NO: 125(Antibody B4).

The present invention provides an anti-IL-36R antibody comprising alight chain comprising the amino acid sequence of SEQ ID NO: 118; and aheavy chain comprising the amino acid sequence of SEQ ID NO: 126(Antibody B5).

The present invention provides an anti-IL-36R antibody comprising alight chain comprising the amino acid sequence of SEQ ID NO: 118; and aheavy chain comprising the amino acid sequence of SEQ ID NO: 127Antibody B6).

The present invention provides an anti-IL-36R antibody comprising alight chain comprising the amino acid sequence of SEQ ID NO: 123; and aheavy chain comprising the amino acid sequence of SEQ ID NO: 138(Antibody C3).

The present invention provides an anti-IL-36R antibody comprising alight chain comprising the amino acid sequence of SEQ ID NO: 123; and aheavy chain comprising the amino acid sequence of SEQ ID NO: 139(Antibody C2).

The present invention provides an anti-IL-36R antibody comprising alight chain comprising the amino acid sequence of SEQ ID NO: 124; and aheavy chain comprising the amino acid sequence of SEQ ID NO: 138(Antibody C1)

Representative antibodies of the present invention are shown below.

TABLE A Anti- body Light Chain Sequences Heavy Chain Sequences B1EIVLTQSPGTLSLSPGERATMSCTA QVQLVQSGAEVKKPGASVKVSCKASGYSSSSVSSSYFHWYQQKPGQAPRLLI FTSSWIHWVRQAPGQGLEWIGEINPGNVYRTSILASGVPDRFSGSGSGTDFTL RTNYNENFRNKATMTVDTSISTAYMELSTISRLEPEDFATYYCHQFHRSPLTF RLRSDDTAVYYCAVVFYGEPYFPYWGQGQGTKLEIKRTVAAPSVFIFPPSD GTLVTVSSASTKGPSVFPLAPSSKSTSGGTEQLKSGTASVVCLLNNFYPREAK AALGCLVKDYFPEPVTVSWNSGALTSGVVQWKVDNALQSGNSQESVTEQD HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTSKDSTYSLSSTLTLSKADYEKHKV YICNVNHKPSNTKVDKRVEPKSCDKTHTYACEVTHQGLSSPVTKSFNRGEC CPPCPAPEAAGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 115)TPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 125) B2 EIVLTQSPGTLSLSPGERATMSCTAQVQLVQSGAEVKKPGASVKVSCKASGYS SSSVSSSYFHWYQQKPGQAPRLLIFTSSWIHWVRQRPGQGLEWIGEINPGNVR YRTSILASGVPDRFSGSGSGTDFTLTNYNENFRNRVTMTVDTSISTAYMELSR TISRLEPEDFATYYCHQFHRSPLTFLRSDDTAVYYCTVVFYGEPYFPYWGQGT GQGTKLEIKRTVAAPSVFIFPPSDLVTVSSASTKGPSVFPLAPSSKSTSGGTAA EQLKSGTASVVCLLNNFYPREAKLGCLVKDYFPEPVTVSWNSGALTSGVHT VQWKVDNALQSGNSQESVTEQDFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI SKDSTYSLSSTLTLSKADYEKHKVCNVNHKPSNTKVDKRVEPKSCDKTHTCP YACEVTHQGLSSPVTKSFNRGECPCPAPEAAGGPSVFLFPPKPKDTLMISRTP (SEQ ID NO: 115)EVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 126) B3 EIVLTQSPGTLSLSPGERATMSCTAQVQLVQSGAEVKKPGASVKVSCKASGYS SSSVSSSYFHWYQQKPGQAPRLLIFTSSWIHWVKQAPGQGLEWMGEINPGNV YRTSILASGVPDRFSGSGSGTDFTLRTNYNENFRNKVTMTVDTSISTAYMELS TISRLEPEDFATYYCHQFHRSPLTFRLRSDDTAVYYCTVVFYGEPYFPYWGQG GQGTKLEIKRTVAAPSVFIFPPSDTLVTVSSASTKGPSVFPLAPSSKSTSGGTA EQLKSGTASVVCLLNNFYPREAKALGCLVKDYFPEPVTVSWNSGALTSGVH VQWKVDNALQSGNSQESVTEQDTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY SKDSTYSLSSTLTLSKADYEKHKVICNVNHKPSNTKVDKRVEPKSCDKTHTC YACEVTHQGLSSPVTKSFNRGECPPCPAPEAAGGPSVFLFPPKPKDTLMISRT (SEQ ID NO: 115)PEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 127) B4 QIVLTQSPGTLSLSPGERATMTCTAQVQLVQSGAEVKKPGASVKVSCKASGYS SSSVSSSYFHWYQQKPGQAPRLWIFTSSWIHWVRQAPGQGLEWIGEINPGNV YRTSRLASGVPDRFSGSGSGTDFTRTNYNENFRNKATMTVDTSISTAYMELS LTISRLEPEDAATYYCHQFHRSPLTRLRSDDTAVYYCAVVFYGEPYFPYWGQ FGAGTKLEIKRTVAAPSVFIFPPSDGTLVTVSSASTKGPSVFPLAPSSKSTSGGT EQLKSGTASVVCLLNNFYPREAKAALGCLVKDYFPEPVTVSWNSGALTSGV VQWKVDNALQSGNSQESVTEQDHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT SKDSTYSLSSTLTLSKADYEKHKVYICNVNHKPSNTKVDKRVEPKSCDKTHT YACEVTHQGLSSPVTKSFNRGECCPPCPAPEAAGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 118)TPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 125) B5 QIVLTQSPGTLSLSPGERATMTCTAQVQLVQSGAEVKKPGASVKVSCKASGYS SSSVSSSYFHWYQQKPGQAPRLWIFTSSWIHWVRQRPGQGLEWIGEINPGNVR YRTSRLASGVPDRFSGSGSGTDFTTNYNENFRNRVTMTVDTSISTAYMELSR LTISRLEPEDAATYYCHQFHRSPLTLRSDDTAVYYCTVVFYGEPYFPYWGQGT FGAGTKLEIKRTVAAPSVFIFPPSDLVTVSSASTKGPSVFPLAPSSKSTSGGTAA EQLKSGTASVVCLLNNFYPREAKLGCLVKDYFPEPVTVSWNSGALTSGVHT VQWKVDNALQSGNSQESVTEQDFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI SKDSTYSLSSTLTLSKADYEKHKVCNVNHKPSNTKVDKRVEPKSCDKTHTCP YACEVTHQGLSSPVTKSFNRGECPCPAPEAAGGPSVFLFPPKPKDTLMISRTP (SEQ ID NO: 118)EVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 126) B6 QIVLTQSPGTLSLSPGERATMTCTAQVQLVQSGAEVKKPGASVKVSCKASGYS SSSVSSSYFHWYQQKPGQAPRLWIFTSSWIHWVKQAPGQGLEWMGEINPGNV YRTSRLASGVPDRFSGSGSGTDFTRTNYNENFRNKVTMTVDTSISTAYMELS LTISRLEPEDAATYYCHQFHRSPLTRLRSDDTAVYYCTVVFYGEPYFPYWGQG FGAGTKLEIKRTVAAPSVFIFPPSDTLVTVSSASTKGPSVFPLAPSSKSTSGGTA EQLKSGTASVVCLLNNFYPREAKALGCLVKDYFPEPVTVSWNSGALTSGVH VQWKVDNALQSGNSQESVTEQDTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY SKDSTYSLSSTLTLSKADYEKHKVICNVNHKPSNTKVDKRVEPKSCDKTHTC YACEVTHQGLSSPVTKSFNRGECPPCPAPEAAGGPSVFLFPPKPKDTLMISRT (SEQ ID NO: 118)PEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 127)

TABLE B Anti body Light Chain Sequences Heavy Chain Sequences C1EIVMTQSPATLSVSPGVRATLSCK QVQLQESGPGLVAPSETLSLTCTVSGFSLASQDVGTNVLWYQQKPGQAPRPL TDYAVHWIRQFPGKGLEWIGVIWSDGSTIYSASYRHSGIPARFSGSGSGTEFTL DFNAPFKSRVTISKDTSKNQVSFKLSSVTTTISSLQSEDFAEYYCQQYSRYPLTF DDTAVYYCARKGGYSGSWFAYWGQGTLGQGTKLEIKRTVAAPSVFIFPPSDE VTVSSASTKGPSVFPLAPSSKSTSGGTAALQLKSGTASVVCLLNNFYPREAKV GCLVKDYFPEPVTVSWNSGALTSGVHTFQWKVDNALQSGNSQESVTEQDSK PAVLQSSGLYSLSSVVTVPSSSLGTQTYICDSTYSLSSTLTLSKADYEKHKVYA NVNHKPSNTKVDKRVEPKSCDKTHTCPPCEVTHQGLSSPVTKSFNRGEC CPAPEAAGGPSVFLFPPKPKDTLMISRTPE (SEQ ID NO: 124)VTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 138) C2 EIVMTQSPATLSVSPGVRATLSCKQVQLQESGPGLVKPSETLSITCTVSGFSLT ASQDVGTNVLWYQQKPGQAPRPLDYAVHWIRQPPGKGLEWIGVIWSDGSTD IYSASYRHSGIPDRFSGSGSGTEFTLYNAPFKSRVTISKDNSKSQVSFKMSSVTA TISSLQSEDFAVYYCQQYSRYPLTFDDTAVYYCARKGGYSGSWFAYWGQGTL GQGTKLEIKRTVAAPSVFIFPPSDEVTVSSASTKGPSVFPLAPSSKSTSGGTAAL QLKSGTASVVCLLNNFYPREAKVGCLVKDYFPEPVTVSWNSGALTSGVHTF QWKVDNALQSGNSQESVTEQDSKPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC DSTYSLSSTLTLSKADYEKHKVYANVNHKPSNTKVDKRVEPKSCDKTHTCPP CEVTHQGLSSPVTKSFNRGECCPAPEAAGGPSVFLFPPKPKDTLMISRTPE (SEQ ID NO: 123)VTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 139) C3 EIVMTQSPATLSVSPGVRATLSCKQVQLQESGPGLVAPSETLSLTCTVSGFSL ASQDVGTNVLWYQQKPGQAPRPLTDYAVHWIRQFPGKGLEWIGVIWSDGST IYSASYRHSGIPDRFSGSGSGTEFTLDFNAPFKSRVTISKDTSKNQVSFKLSSVTT TISSLQSEDFAVYYCQQYSRYPLTFDDTAVYYCARKGGYSGSWFAYWGQGTL GQGTKLEIKRTVAAPSVFIFPPSDEVTVSSASTKGPSVFPLAPSSKSTSGGTAAL QLKSGTASVVCLLNNFYPREAKVGCLVKDYFPEPVTVSWNSGALTSGVHTF QWKVDNALQSGNSQESVTEQDSKPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC DSTYSLSSTLTLSKADYEKHKVYANVNHKPSNTKVDKRVEPKSCDKTHTCPP CEVTHQGLSSPVTKSFNRGECCPAPEAAGGPSVFLFPPKPKDTLMISRTPE (SEQ ID NO: 123)VTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 138)

The antibodies of the present invention are useful in methods for thetreatment of various diseases or disorders, for example immunological,inflammatory, autoimmune diseases and respiratory diseases in humans.For example, the antibodies of the present invention are useful inmethods for the treatment of psoriasis, rheumatoid arthritis,inflammatory bowel disease or psoriatic arthritis. For example, theantibodies of the present invention are useful in methods for thetreatment of chronic obstructive pulmonary disorder (COPD) or asthma.For example, the antibodies of the present invention are useful inmethods for the treatment of scleroderma, palmoplantar pustulosis,generalized pustular psoriasis, diabetic nephropathy, lupus nephritis,scleroderma, ankylosing spondylitis, deficiency in the IL-36 receptorantagonist autoimmune disease (DITRA), deficiency in the IL-1 receptorantagonist autoimmune disease (DIRA) or cryopyrin associated periodicsyndromes (CAPS).

In some aspects, the humanized antibody displays blocking activity,whereby it decreases the binding of IL-36 ligand to IL-36 receptor by atleast 45%, by at least 50%, by at least 55%, by at least 60%, by atleast 65%, by at least 70%, by at least 75%, by at least 80%, by atleast 85%, by at least 90%, or by at least 95%. The ability of anantibody to block binding of IL-36 ligand to the IL-36 receptor can bemeasured using competitive binding assays known in the art.Alternatively, the blocking activity of an antibody can be measured byassessing the biological effects of IL-36, such as the production ofIL-8, IL-6, and GM-CSF to determine if signaling mediated by the IL-36receptor is inhibited.

In a further aspect, the present invention provides a humanizedanti-IL-36R antibody having favorable biophysical properties. In oneaspect, a humanized anti-IL-36R antibody of the present invention ispresent in at least 90% monomer form, or in at least 92% monomer form,or in at least 95% monomer form in a buffer. In a further aspect, ahumanized anti-IL-36R antibody of the present invention remains in atleast 90% monomer form, or in at least 92% monomer form, or in at least95% monomer form in a buffer for one month or for four months.

In one aspect, a humanized antibody of the present invention is AntibodyB1, Antibody B2, Antibody B3, Antibody B4, Antibody B5, Antibody B6,Antibody C1, Antibody C2, or Antibody C3. Accordingly, in oneembodiment, a humanized antibody of the present invention comprises thelight chain sequence of SEQ ID NO:115 and the heavy chain sequence ofSEQ ID NO:125 (Antibody B1). In another embodiment, a humanized antibodyof the present invention comprises the light chain sequence of SEQ IDNO:115 and the heavy chain sequence of SEQ ID NO:126 (Antibody B2). Inanother embodiment, a humanized antibody of the present inventioncomprises the light chain sequence of SEQ ID NO:115 and the heavy chainsequence of SEQ ID NO:127 (Antibody B3). In another embodiment, ahumanized antibody of the present invention comprises the light chainsequence of SEQ ID NO:118 and the heavy chain sequence of SEQ ID NO:125(Antibody B4). In another embodiment, a humanized antibody of thepresent invention comprises the light chain sequence of SEQ ID NO:118and the heavy chain sequence of SEQ ID NO:126 (Antibody B5). In anotherembodiment, a humanized antibody of the present invention comprises thelight chain sequence of SEQ ID NO:118 and the heavy chain sequence ofSEQ ID NO:127 (Antibody B6). In another embodiment, a humanized antibodyof the present invention comprises the light chain sequence of SEQ IDNO:124 and the heavy chain sequence of SEQ ID NO:138 (Antibody C1). Inanother embodiment, a humanized antibody of the present inventioncomprises the light chain sequence of SEQ ID NO:123 and the heavy chainsequence of SEQ ID NO:139 (Antibody C2). In another embodiment, ahumanized antibody of the present invention comprises the light chainsequence of SEQ ID NO:123 and the heavy chain sequence of SEQ ID NO:138(Antibody C3).

In a further embodiment, a humanized antibody of the present inventionconsists of the light chain sequence of SEQ ID NO:115 and the heavychain sequence of SEQ ID NO:125 (Antibody B1). In another embodiment, ahumanized antibody of the present invention consists of the light chainsequence of SEQ ID NO:115 and the heavy chain sequence of SEQ ID NO:126(Antibody B2). In another embodiment, a humanized antibody of thepresent invention consists of the light chain sequence of SEQ ID NO:115and the heavy chain sequence of SEQ ID NO:127 (Antibody B3). In anotherembodiment, a humanized antibody of the present invention consists ofthe light chain sequence of SEQ ID NO:118 and the heavy chain sequenceof SEQ ID NO:125 (Antibody B4). In another embodiment, a humanizedantibody of the present invention consists of the light chain sequenceof SEQ ID NO:118 and the heavy chain sequence of SEQ ID NO:126 (AntibodyB5). In another embodiment, a humanized antibody of the presentinvention consists of the light chain sequence of SEQ ID NO:118 and theheavy chain sequence of SEQ ID NO:127 (Antibody B6). In anotherembodiment, a humanized antibody of the present invention consists ofthe light chain sequence of SEQ ID NO:124 and the heavy chain sequenceof SEQ ID NO:138 (Antibody C1). In another embodiment, a humanizedantibody of the present invention consists of the light chain sequenceof SEQ ID NO:123 and the heavy chain sequence of SEQ ID NO:139 (AntibodyC2). In another embodiment, a humanized antibody of the presentinvention consists of the light chain sequence of SEQ ID NO:123 and theheavy chain sequence of SEQ ID NO:138 (Antibody C3).

In some embodiments, the humanized anti-IL-36R antibodies, includingantigen-binding fragments thereof, such as heavy and light chainvariable regions, comprise an amino acid sequence of the residuesderived from Antibody B1, Antibody B2, Antibody B3, Antibody B4,Antibody B5, Antibody B6, Antibody C1, Antibody C2, or Antibody C3.

In a further embodiment, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof that competitively binds tohuman IL-36R with an antibody of the present invention, for exampleAntibody B1, Antibody B2, Antibody B3, Antibody B4, Antibody B5,Antibody B6, Antibody C1, Antibody C2 or Antibody C3 described herein.The ability of an antibody or antigen-binding fragment to competitivelybind to IL-36R can be measured using competitive binding assays known inthe art.

The humanized anti-IL-36R antibodies optionally include specific aminoacid substitutions in the consensus or germline framework regions. Thespecific substitution of amino acid residues in these frameworkpositions can improve various aspects of antibody performance includingbinding affinity and/or stability, over that demonstrated in humanizedantibodies formed by “direct swap” of CDRs or HVLs into the humangermline framework regions.

In some embodiments, the present invention describes other monoclonalantibodies with a light chain variable region having the amino acidsequence set forth in any one of SEQ ID NO:1-10. In some embodiments,the present invention describes other monoclonal antibodies with a heavychain variable region having the amino acid sequence set forth in anyone of SEQ ID NO:11-20. Placing such CDRs into FRs of the humanconsensus heavy and light chain variable domains will yield usefulhumanized antibodies of the present invention.

In particular, the present invention provides monoclonal antibodies withthe combinations of light chain variable and heavy chain variableregions of SEQ ID NO:1/11, 2/12, 3/13, 4/14, 5/15, 6/16, 7/17, 8/18,9/19, 10/20. Such variable regions can be combined with human constantregions.

In some embodiments, the present invention describes other humanizedantibodies with light chain variable region sequences having the aminoacid sequence set forth in any one of SEQ ID NO:76-86. In someembodiments, the present invention describes other humanized antibodieswith heavy chain variable region sequences having the amino acidsequence set forth in any one of SEQ ID NO:87-101. In particular, thepresent invention provides monoclonal antibodies with the combinationsof light chain variable and heavy chain variable regions of SEQ ID NO:77/89, 80/88, 80/89, 77/87, 77/88, 80/87, 86/100, 85/101, 85/100. Suchvariable regions can be combined with human constant regions.

In a further embodiment, the present invention relates to an anti-IL-36Rantibody or antigen-binding fragment thereof comprising a humanizedlight chain variable domain comprising the CDRs of SEQ ID NO:77 andframework regions having an amino acid sequence at least 90% identical,at least 93% identical or at least 95% identical to the amino acidsequence of the framework regions of the variable domain light chainamino acid sequence of SEQ ID NO:77 and a humanized heavy chain variabledomain comprising the CDRs of SEQ ID NO:89 and framework regions havingan amino acid sequence at least 90% identical, at least 93% identical orat least 95% identical to the amino acid sequence of the frameworkregions of the variable domain heavy chain amino acid sequence of SEQ IDNO:89. In one embodiment, the anti-IL-36R antibody is a humanizedmonoclonal antibody.

In a further embodiment, the present invention relates to an anti-IL-36Rantibody or antigen-binding fragment thereof comprising a humanizedlight chain variable domain comprising the CDRs of SEQ ID NO:80 andframework regions having an amino acid sequence at least 90% identical,at least 93% identical or at least 95% identical to the amino acidsequence of the framework regions of the variable domain light chainamino acid sequence of SEQ ID NO:80 and a humanized heavy chain variabledomain comprising the CDRs of SEQ ID NO:88 and framework regions havingan amino acid sequence at least 90% identical, at least 93% identical orat least 95% identical to the amino acid sequence of the frameworkregions of the variable domain heavy chain amino acid sequence of SEQ IDNO:88. In one embodiment, the anti-IL-36R antibody is a humanizedmonoclonal antibody.

In a further embodiment, the present invention relates to an anti-IL-36Rantibody or antigen-binding fragment thereof comprising a humanizedlight chain variable domain comprising the CDRs of SEQ ID NO:80 andframework regions having an amino acid sequence at least 90% identical,at least 93% identical or at least 95% identical to the amino acidsequence of the framework regions of the variable domain light chainamino acid sequence of SEQ ID NO:80 and a humanized heavy chain variabledomain comprising the CDRs of SEQ ID NO:89 and framework regions havingan amino acid sequence at least 90% identical, at least 93% identical orat least 95% identical to the amino acid sequence of the frameworkregions of the variable domain heavy chain amino acid sequence of SEQ IDNO:89. In one embodiment, the anti-IL-36R antibody is a humanizedmonoclonal antibody.

In a further embodiment, the present invention relates to an anti-IL-36Rantibody or antigen-binding fragment thereof comprising a humanizedlight chain variable domain comprising the CDRs of SEQ ID NO:77 andframework regions having an amino acid sequence at least 90% identical,at least 93% identical or at least 95% identical to the amino acidsequence of the framework regions of the variable domain light chainamino acid sequence of SEQ ID NO:77 and a humanized heavy chain variabledomain comprising the CDRs of SEQ ID NO:87 and framework regions havingan amino acid sequence at least 90% identical, at least 93% identical orat least 95% identical to the amino acid sequence of the frameworkregions of the variable domain heavy chain amino acid sequence of SEQ IDNO:87. In one embodiment, the anti-IL-36R antibody is a humanizedmonoclonal antibody.

In a further embodiment, the present invention relates to an anti-IL-36Rantibody or antigen-binding fragment thereof comprising a humanizedlight chain variable domain comprising the CDRs of SEQ ID NO:77 andframework regions having an amino acid sequence at least 90% identical,at least 93% identical or at least 95% identical to the amino acidsequence of the framework regions of the variable domain light chainamino acid sequence of SEQ ID NO:77 and a humanized heavy chain variabledomain comprising the CDRs of SEQ ID NO:88 and framework regions havingan amino acid sequence at least 90% identical, at least 93% identical orat least 95% identical to the amino acid sequence of the frameworkregions of the variable domain heavy chain amino acid sequence of SEQ IDNO:88. In one embodiment, the anti-IL-36R antibody is a humanizedmonoclonal antibody.

In a further embodiment, the present invention relates to an anti-IL-36Rantibody or antigen-binding fragment thereof comprising a humanizedlight chain variable domain comprising the CDRs of SEQ ID NO:80 andframework regions having an amino acid sequence at least 90% identical,at least 93% identical or at least 95% identical to the amino acidsequence of the framework regions of the variable domain light chainamino acid sequence of SEQ ID NO:80 and a humanized heavy chain variabledomain comprising the CDRs of SEQ ID NO:87 and framework regions havingan amino acid sequence at least 90% identical, at least 93% identical orat least 95% identical to the amino acid sequence of the frameworkregions of the variable domain heavy chain amino acid sequence of SEQ IDNO:87. In one embodiment, the anti-IL-36R antibody is a humanizedmonoclonal antibody.

In a further embodiment, the present invention relates to an anti-IL-36Rantibody or antigen-binding fragment thereof comprising a humanizedlight chain variable domain comprising the CDRs of SEQ ID NO:86 andframework regions having an amino acid sequence at least 90% identical,at least 93% identical or at least 95% identical to the amino acidsequence of the framework regions of the variable domain light chainamino acid sequence of SEQ ID NO:86 and a humanized heavy chain variabledomain comprising the CDRs of SEQ ID NO:100 and framework regions havingan amino acid sequence at least 90% identical, at least 93% identical orat least 95% identical to the amino acid sequence of the frameworkregions of the variable domain heavy chain amino acid sequence of SEQ IDNO:100. In one embodiment, the anti-IL-36R antibody is a humanizedmonoclonal antibody.

In a further embodiment, the present invention relates to an anti-IL-36Rantibody or antigen-binding fragment thereof comprising a humanizedlight chain variable domain comprising the CDRs of SEQ ID NO:85 andframework regions having an amino acid sequence at least 90% identical,at least 93% identical or at least 95% identical to the amino acidsequence of the framework regions of the variable domain light chainamino acid sequence of SEQ ID NO:85 and a humanized heavy chain variabledomain comprising the CDRs of SEQ ID NO:101 and framework regions havingan amino acid sequence at least 90% identical, at least 93% identical orat least 95% identical to the amino acid sequence of the frameworkregions of the variable domain heavy chain amino acid sequence of SEQ IDNO:101. In one embodiment, the anti-IL-36R antibody is a humanizedmonoclonal antibody.

In a further embodiment, the present invention relates to an anti-IL-36Rantibody or antigen-binding fragment thereof comprising a humanizedlight chain variable domain comprising the CDRs of SEQ ID NO:85 andframework regions having an amino acid sequence at least 90% identical,at least 93% identical or at least 95% identical to the amino acidsequence of the framework regions of the variable domain light chainamino acid sequence of SEQ ID NO:85 and a humanized heavy chain variabledomain comprising the CDRs of SEQ ID NO:100 and framework regions havingan amino acid sequence at least 90% identical, at least 93% identical orat least 95% identical to the amino acid sequence of the frameworkregions of the variable domain heavy chain amino acid sequence of SEQ IDNO:100. In one embodiment, the anti-IL-36R antibody is a humanizedmonoclonal antibody.

In some specific embodiments, the humanized anti-IL-36R antibodiesdisclosed herein comprise at least a heavy or a light chain variabledomain comprising the CDRs or HVLs of the murine monoclonal antibodiesor humanized antibodies as disclosed herein and the FRs of the humangermline heavy and light chain variable domains.

In one further aspect, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof comprising a light chainCDR1 (L-CDR1) sequence of any one of SEQ ID NO:21-29; a light chain CDR2(L-CDR2) sequence of any one of SEQ ID NO:30-38; a light chain CDR3(L-CDR3) sequence of any one of SEQ ID NO:39-47; a heavy chain CDR1(H-CDR1) sequence of any one of SEQ ID NO:48-56; a heavy chain CDR2(H-CDR2) sequence of any one of SEQ ID NO:57-66; and a heavy chain CDR3(H-CDR3) sequence of any one of SEQ ID NO:67-75. In one aspect, theanti-IL-36R antibody or antigen-binding fragment thereof comprises alight chain variable region comprising a L-CDR1 listed above, a L-CDR2listed above and a L-CDR3 listed above, and a heavy chain variableregion comprising a H-CDR1 listed above, a H-CDR2 listed above and aH-CDR3 listed above.

In a further aspect, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof comprising:

-   -   a) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3        sequence of SEQ ID NO:21, 30, 39, 48, 57 and 67, respectively;        or    -   b) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3        sequence of SEQ ID NO:22, 31, 40, 49, 58 and 68, respectively;        or    -   c) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3        sequence of SEQ ID NO:23, 32, 41, 50, 59 and 69, respectively;        or    -   d) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3        sequence of SEQ ID NO:24, 33, 42, 51, 60 and 70, respectively;        or    -   e) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3        sequence of SEQ ID NO:25, 34, 43, 52, 61 and 71, respectively;        or    -   f) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3        sequence of SEQ ID NO:26, 35, 44, 53, 62 and 72, respectively;        or    -   g) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3        sequence of SEQ ID NO:27, 36, 45, 54, 63 and 73, respectively;        or    -   h) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3        sequence of SEQ ID NO:27, 36, 45, 54, 64 and 74, respectively;        or    -   i) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3        sequence of SEQ ID NO:27, 36, 45, 54, 64 and 73, respectively;        or    -   j) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3        sequence of SEQ ID NO:28, 37, 46, 55, 65 and 74, respectively;        or    -   k) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3        sequence of SEQ ID NO:29, 38, 47, 56, 66 and 75, respectively.

In a further aspect, the present invention provides an anti-IL-36Rantibody or antigen-binding fragment thereof comprising:

-   -   a) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3        sequence of SEQ ID NO:26, 103, 44, 53, 62 and 72, respectively;        or    -   b) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3        sequence of SEQ ID NO:26, 104, 44, 53, 62 and 72, respectively;        or    -   c) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3        sequence of SEQ ID NO:27, 36, 45, 107, 63 and 73, respectively;        or    -   d) a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3        sequence of SEQ ID NO:27, 36, 45, 107, 64 or 73, respectively.

In one aspect, the anti-IL-36R antibody or antigen-binding fragmentthereof comprises a light chain variable region comprising a L-CDR1,L-CDR2 and L-CDR3 combination listed above, and a heavy chain variableregion comprising a H-CDR1, H-CDR2 and H-CDR3 combination listed above.

In specific embodiments, it is contemplated that chimeric antibodieswith switched CDR regions (i.e., for example switching one or two CDRsof one of the mouse antibodies or humanized antibody derived therefromwith the analogous CDR from another mouse antibody or humanized antibodyderived therefrom) between these exemplary immunoglobulins may yielduseful antibodies.

In certain embodiments, the humanized anti-IL-36R antibody is anantibody fragment. Various antibody fragments have been generallydiscussed above and there are techniques that have been developed forthe production of antibody fragments. Fragments can be derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,1992, Journal of Biochemical and Biophysical Methods 24:107-117; andBrennan et al., 1985, Science 229:81). Alternatively, the fragments canbe produced directly in recombinant host cells. For example, Fab′-SHfragments can be directly recovered from E. coli and chemically coupledto form F(ab′)₂ fragments (see, e.g., Carter et al., 1992,Bio/Technology 10:163-167). By another approach, F(ab′)₂ fragments canbe isolated directly from recombinant host cell culture. Othertechniques for the production of antibody fragments will be apparent tothe skilled practitioner.

Accordingly, in one aspect, the present invention provides antibodyfragments comprising the CDRs described herein, in particular one of thecombinations of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3described herein. In a further aspect, the present invention providesantibody fragments comprising the variable regions described herein, forexample one of the combinations of light chain variable regions andheavy chain variable regions described herein.

Certain embodiments include an F(ab′)₂ fragment of a humanizedanti-IL-36R antibody comprise a light chain sequence of any of SEQ IDNO: 115 or 118 in combination with a heavy chain sequence of SEQ ID NO:125, 126 or 127. Such embodiments can include an intact antibodycomprising such an F(ab′)₂.

Certain embodiments include an F(ab′)₂ fragment of a humanizedanti-IL-36R antibody comprise a light chain sequence of any of SEQ IDNO: 123 or 124 in combination with a heavy chain sequence of SEQ ID NO:138 or 139. Such embodiments can include an intact antibody comprisingsuch an F(ab′)₂.

In some embodiments, the antibody or antibody fragment includes aconstant region that mediates effector function. The constant region canprovide antibody-dependent cellular cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP) and/orcomplement-dependent cytotoxicity (CDC) responses against an IL-36Rexpressing target cell. The effector domain(s) can be, for example, anFc region of an Ig molecule.

The effector domain of an antibody can be from any suitable vertebrateanimal species and isotypes. The isotypes from different animal speciesdiffer in the abilities to mediate effector functions. For example, theability of human immunoglobulin to mediate CDC and ADCC/ADCP isgenerally in the order of IgM≈IgG₁≈IgG₃>IgG₂>IgG₄ andIgG₁≈IgG₃>IgG₂/IgM/Iga₄, respectively. Murine immunoglobulins mediateCDC and ADCC/ADCP generally in the order of murineIgM≈IgG₃>>IgG_(2b)>IgG_(2a)>>IgG₁ and IgG_(2b)>IgG_(2a)>IgG₁>>IgG₃,respectively. In another example, murine IgG_(2a) mediates ADCC whileboth murine IgG_(2a) and IgM mediate CDC.

Antibody Modifications

The humanized anti-IL-36R antibodies and agents can includemodifications of the humanized anti-IL-36R antibody or antigen-bindingfragment thereof. For example, it may be desirable to modify theantibody with respect to effector function, so as to enhance theeffectiveness of the antibody in treating cancer. One such modificationis the introduction of cysteine residue(s) into the Fc region, therebyallowing interchain disulfide bond formation in this region. Thehomodimeric antibody thus generated can have improved internalizationcapability and/or increased complement-mediated cell killing and/orantibody-dependent cellular cytotoxicity (ADCC). See, for example, Caronet al., 1992, J. Exp Med. 176:1191-1195; and Shopes, 1992, J. Immunol.148:2918-2922. Homodimeric antibodies having enhanced anti-tumoractivity can also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al., 1993, Cancer Research 53: 2560-2565.Alternatively, an antibody can be engineered to contain dual Fc regions,enhancing complement lysis and ADCC capabilities of the antibody. SeeStevenson et al., 1989, Anti-Cancer Drug Design 3: 219-230.

Antibodies with improved ability to support ADCC have been generated bymodifying the glycosylation pattern of their Fc region. This is possiblesince antibody glycosylation at the asparagine residue, N297, in theC_(H2) domain is involved in the interaction between IgG and Fcγreceptors prerequisite to ADCC. Host cell lines have been engineered toexpress antibodies with altered glycosylation, such as increasedbisecting N-acetylglucosamine or reduced fucose. Fucose reductionprovides greater enhancement to ADCC activity than does increasing thepresence of bisecting N-acetylglucosamine. Moreover, enhancement of ADCCby low fucose antibodies is independent of the FcγRIIIa V/Fpolymorphism.

Modifying the amino acid sequence of the Fc region of antibodies is analternative to glycosylation engineering to enhance ADCC. The bindingsite on human IgG₁ for Fcγ receptors has been determined by extensivemutational analysis. This led to the generation of humanized IgG₁antibodies with Fc mutations that increase the binding affinity forFcγRIIIa and enhance ADCC in vitro. Additionally, Fc variants have beenobtained with many different permutations of binding properties, e.g.,improved binding to specific FcγR receptors with unchanged or diminishedbinding to other FcγR receptors.

Another aspect includes immunoconjugates comprising the humanizedantibody or fragments thereof conjugated to a cytotoxic agent such as achemotherapeutic agent, a toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used to form useful immunoconjugatesinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, the tricothecenes, and the like. A variety of radionuclidesare available for the production of radioconjugated humanizedanti-IL-36R antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and¹⁸⁶Re.

Conjugates of the humanized anti-IL-36R antibody and cytotoxic orchemotherapeutic agent can be made by known methods, using a variety ofbifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., 1987, Science 238:1098.Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. Conjugates also can beformed with a cleavable linker.

The humanized anti-IL-36R antibodies disclosed herein can also beformulated as immunoliposomes. Liposomes containing the antibody areprepared by methods known in the art, such as described in Epstein etal., 1985, Proc. Natl. Acad. Sci. USA 82:3688; Hwang et al., 1980, Proc.Natl. Acad. Sci. USA 77:4030; and U.S. Pat. Nos. 4,485,045 and4,544,545. Liposomes having enhanced circulation time are disclosed, forexample, in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of an antibody disclosed herein can beconjugated to the liposomes as described in Martin et al., 1982, J.Biol. Chem. 257:286-288 via a disulfide interchange reaction. Achemotherapeutic agent (such as doxorubicin) is optionally containedwithin the liposome. See, e.g., Gabizon et al., 1989, J. National CancerInst. 81(19):1484.

The antibodies described and disclosed herein can also be used in ADEPT(Antibody-Directed Enzyme Prodrug Therapy) procedures by conjugating theantibody to a prodrug-activating enzyme that converts a prodrug (e.g., apeptidyl chemotherapeutic agent), to an active anti-cancer drug. See,for example, WO 81/01145, WO 88/07378, and U.S. Pat. No. 4,975,278. Theenzyme component of the immunoconjugate useful for ADEPT is an enzymecapable of acting on a prodrug in such a way so as to covert it into itsmore active, cytotoxic form. Specific enzymes that are useful in ADEPTinclude, but are not limited to, alkaline phosphatase for convertingphosphate-containing prodrugs into free drugs; arylsulfatase forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase for converting non-toxic 5-fluorocytosine into the anti-cancerdrug, 5-fluorouracil; proteases, such as serratia protease, thermolysin,subtilisin, carboxypeptidases, and cathepsins (such as cathepsins B andL), for converting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, for converting prodrugs containing D-aminoacid substituents; carbohydrate-cleaving enzymes such as β-galactosidaseand neuraminidase for converting glycosylated prodrugs into free drugs;β-lactamase for converting drugs derivatized with β-lactams into freedrugs; and penicillin amidases, such as penicillin V amidase orpenicillin G amidase, for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies having enzymatic activity(“abzymes”) can be used to convert the prodrugs into free active drugs(see, for example, Massey, 1987, Nature 328: 457-458). Antibody-abzymeconjugates can be prepared by known methods for delivery of the abzymeto a tumor cell population, for example, by covalently binding theenzyme to the humanized anti-IL-36R antibody/heterobifunctionalcrosslinking reagents discussed above. Alternatively, fusion proteinscomprising at least the antigen binding region of an antibody disclosedherein linked to at least a functionally active portion of an enzyme asdescribed above can be constructed using recombinant DNA techniques(see, e.g., Neuberger et al., 1984, Nature 312:604-608).

In certain embodiments, it may be desirable to use a humanizedanti-IL-36R antibody fragment, rather than an intact antibody, toincrease tissue penetration, for example. It may be desirable to modifythe antibody fragment in order to increase its serum half life. This canbe achieved, for example, by incorporation of a salvage receptor bindingepitope into the antibody fragment. In one method, the appropriateregion of the antibody fragment can be altered (e.g., mutated), or theepitope can be incorporated into a peptide tag that is then fused to theantibody fragment at either end or in the middle, for example, by DNA orpeptide synthesis. See, e.g., WO 96/32478.

In other embodiments, covalent modifications of the humanizedanti-IL-36R antibody are also included. Covalent modifications includemodification of cysteinyl residues, histidyl residues, lysinyl andamino-terminal residues, arginyl residues, tyrosyl residues, carboxylside groups (aspartyl or glutamyl), glutaminyl and asparaginyl residues,or seryl, or threonyl residues. Another type of covalent modificationinvolves chemically or enzymatically coupling glycosides to theantibody. Such modifications may be made by chemical synthesis or byenzymatic or chemical cleavage of the antibody, if applicable. Othertypes of covalent modifications of the antibody can be introduced intothe molecule by reacting targeted amino acid residues of the antibodywith an organic derivatizing agent that is capable of reacting withselected side chains or the amino- or carboxy-terminal residues.

Removal of any carbohydrate moieties present on the antibody can beaccomplished chemically or enzymatically. Chemical deglycosylation isdescribed by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 andby Edge et al., 1981, Anal. Biochem., 118:131. Enzymatic cleavage ofcarbohydrate moieties on antibodies can be achieved by the use of avariety of endo- and exo-glycosidases as described by Thotakura et al.,1987, Meth. Enzymol 138:350.

Another type of useful covalent modification comprises linking theantibody to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in one or more of U.S. Pat. No. 4,640,835, U.S. Pat.No. 4,496,689, U.S. Pat. No. 4,301,144, U.S. Pat. No. 4,670,417, U.S.Pat. No. 4,791,192 and U.S. Pat. No. 4,179,337.

Humanization and Amino Acid Sequence Variants

Amino acid sequence variants of the anti-IL-36R antibody can be preparedby introducing appropriate nucleotide changes into the anti-IL-36Rantibody DNA, or by peptide synthesis. Such variants include, forexample, deletions from, and/or insertions into and/or substitutions of,residues within the amino acid sequences of the anti-IL-36R antibodiesof the examples herein. Any combination of deletions, insertions, andsubstitutions is made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid changes also may alter post-translational processes of thehumanized or variant anti-IL-36R antibody, such as changing the numberor position of glycosylation sites.

A useful method for identification of certain residues or regions of theanti-IL-36R antibody that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis,” as described by Cunningham andWells (Science, 244:1081-1085 (1989)). Here, a residue or group oftarget residues are identified (e.g., charged residues such as arg, asp,his, lys, and glu) and replaced by a neutral or negatively charged aminoacid (typically alanine) to affect the interaction of the amino acidswith IL-36R antigen. Those amino acid locations demonstrating functionalsensitivity to the substitutions then are refined by introducing furtheror other variants at, or for, the sites of substitution. Thus, while thesite for introducing an amino acid sequence variation is predetermined,the nature of the mutation per se need not be predetermined. Forexample, to analyze the performance of a mutation at a given site,alanine scanning or random mutagenesis is conducted at the target codonor region and the expressed anti-IL-36R antibody variants are screenedfor the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean anti-IL-36R antibody fused to an epitope tag. Other insertionalvariants of the anti-IL-36R antibody molecule include a fusion to the N-or C-terminus of the anti-IL-36R antibody of an enzyme or a polypeptidewhich increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the anti-IL-36Rantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 5 under the heading of“preferred substitutions”. If such substitutions result in a change inbiological activity, then more substantial changes, denominated“exemplary substitutions”, or as further described below in reference toamino acid classes, may be introduced and the products screened.

TABLE C Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;asp, lys; arg gln Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn;glu asn Glu (E) asp; gln asp Gly (G) ala ala His (H) arg; asn; gln; lys;arg Ile (I) leu; val; met; ala; phe; norleucine leu Leu (L) ile;norleucine; val; met; ala; phe ile Lys (K) arg; gln; asn arg Met (M)leu; phe; ile leu Phe (F) tyr; leu; val; ile; ala; tyr Pro (P) ala alaSer (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) phe; trp;thr; ser phe Val (V) leu; ile; met; phe ala; norleucine; leu

In protein chemistry, it is generally accepted that the biologicalproperties of the antibody can be accomplished by selectingsubstitutions that differ significantly in their effect on maintaining(a) the structure of the polypeptide backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b) thecharge or hydrophobicity of the molecule at the target site, or (c) thebulk of the side chain. Naturally occurring residues are divided intogroups based on common side-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;(2) neutral hydrophilic: cys, ser, thr;(3) acidic: asp, glu;(4) basic: asn, gin, his, lys, arg;(5) residues that influence chain orientation: gly, pro; and(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the humanized or variant anti-IL-36R antibody also may besubstituted, generally with serine, to improve the oxidative stabilityof the molecule, prevent aberrant crosslinking, or provide forestablished points of conjugation to a cytotoxic or cytostatic compound.Conversely, cysteine bond(s) may be added to the antibody to improve itsstability (particularly where the antibody is an antibody fragment suchas an Fv fragment).

A type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g., a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have improved biological properties relative tothe parent antibody from which they are generated. A convenient way forgenerating such substitutional variants is affinity maturation usingphage display. Briefly, several hypervariable region sites (e.g., 6-7sites) are mutated to generate all possible amino substitutions at eachsite. The antibody variants thus generated are displayed in a monovalentfashion from filamentous phage particles as fusions to the gene IIIproduct of M13 packaged within each particle. The phage-displayedvariants are then screened for their biological activity (e.g., bindingaffinity). In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or in addition, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and human IL-36R. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By “altering” is meant deletingone or more carbohydrate moieties found in the antibody, and/or addingone or more glycosylation sites that are not present in the antibody.

In some embodiments, it may be desirable to modify the antibodies of theinvention to add glycosylations sites. Glycosylation of antibodies istypically either N-linked or 0-linked. N-linked refers to the attachmentof the carbohydrate moiety to the side chain of an asparagine residue.The tripeptide sequences asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. Thus, in order to glycosylate a givenprotein, e.g., an antibody, the amino acid sequence of the protein isengineered to contain one or more of the above-described tripeptidesequences (for N-linked glycosylation sites). The alteration may also bemade by the addition of, or substitution by, one or more serine orthreonine residues to the sequence of the original antibody (forO-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of theanti-IL-36R antibody are prepared by a variety of methods known in theart. These methods include, but are not limited to, isolation from anatural source (in the case of naturally occurring amino acid sequencevariants) or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the anti-IL-36R antibody.

Polynucleotides, Vectors, Host Cells, and Recombinant Methods

Other embodiments encompass isolated polynucleotides that comprise asequence encoding a humanized anti-IL-36R antibody, vectors, and hostcells comprising the polynucleotides, and recombinant techniques forproduction of the humanized antibody. The isolated polynucleotides canencode any desired form of the anti-IL-36R antibody including, forexample, full length monoclonal antibodies, Fab, Fab′, F(ab′)₂, and Fvfragments, diabodies, linear antibodies, single-chain antibodymolecules, and multispecific antibodies formed from antibody fragments.

Some embodiments include isolated polynucleotides comprising sequencesthat encode the light chain variable region of an antibody or antibodyfragment having the amino acid sequence of any of SEQ ID NO: SEQ IDNO:1-10. Some embodiments include isolated polynucleotides comprisingsequences that encode the heavy chain variable region of an antibody orantibody fragment having the amino acid sequence of SEQ ID NO:11-20.

Some embodiments include isolated polynucleotides comprising sequencesthat encode the light chain variable region of an antibody or antibodyfragment having the amino acid sequence of any of SEQ ID NO:76-86. Someembodiments include isolated polynucleotides comprising sequences thatencode the heavy chain variable region of an antibody or antibodyfragment having the amino acid sequence of SEQ ID NO: 87-101.

Some embodiments include isolated polynucleotides comprising sequencesthat encode the light chain of an antibody having the amino acidsequence of any of SEQ ID NO:114-124. Some embodiments include isolatedpolynucleotides comprising sequences that encode the heavy chain of anantibody having the amino acid sequence of SEQ ID NO:125-139.

In one aspect, the isolated polynucleotide sequence(s) encodes anantibody or antibody fragment having a light chain and a heavy chainvariable region comprising the amino acid sequences of SEQ ID NO:115 andSEQ ID NO:127, respectively; SEQ ID NO:118 and SEQ ID NO:126,respectively; SEQ ID NO:118 and SEQ ID NO:127, respectively; SEQ IDNO:115 and SEQ ID NO:125, respectively; SEQ ID NO:115 and SEQ ID NO:126,respectively; SEQ ID NO:118 and SEQ ID NO:125, respectively; SEQ IDNO:124 and SEQ ID NO:138, respectively; SEQ ID NO:123 and SEQ ID NO:139,respectively; SEQ ID NO:123 and SEQ ID NO:138, respectively.

The polynucleotide(s) that comprise a sequence encoding a humanizedanti-IL-36R antibody or a fragment or chain thereof can be fused to oneor more regulatory or control sequence, as known in the art, and can becontained in suitable expression vectors or host cell as known in theart. Each of the polynucleotide molecules encoding the heavy or lightchain variable domains can be independently fused to a polynucleotidesequence encoding a constant domain, such as a human constant domain,enabling the production of intact antibodies. Alternatively,polynucleotides, or portions thereof, can be fused together, providing atemplate for production of a single chain antibody.

For recombinant production, a polynucleotide encoding the antibody isinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Many suitable vectors for expressing the recombinantantibody are available. The vector components generally include, but arenot limited to, one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

The humanized anti-IL-36R antibodies can also be produced as fusionpolypeptides, in which the antibody is fused with a heterologouspolypeptide, such as a signal sequence or other polypeptide having aspecific cleavage site at the amino terminus of the mature protein orpolypeptide. The heterologous signal sequence selected is typically onethat is recognized and processed (i.e., cleaved by a signal peptidase)by the host cell. For prokaryotic host cells that do not recognize andprocess the humanized anti-IL-36R antibody signal sequence, the signalsequence can be substituted by a prokaryotic signal sequence. The signalsequence can be, for example, alkaline phosphatase, penicillinase,lipoprotein, heat-stable enterotoxin II leaders, and the like. For yeastsecretion, the native signal sequence can be substituted, for example,with a leader sequence obtained from yeast invertase alpha-factor(including Saccharomyces and Kluyveromyces α-factor leaders), acidphosphatase, C. albicans glucoamylase, or the signal described inWO90/13646. In mammalian cells, mammalian signal sequences as well asviral secretory leaders, for example, the herpes simplex gD signal, canbe used. The DNA for such precursor region is ligated in reading frameto DNA encoding the humanized anti-IL-36R antibody.

Expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2-υ. plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV, andBPV) are useful for cloning vectors in mammalian cells. Generally, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

Expression and cloning vectors may contain a gene that encodes aselectable marker to facilitate identification of expression. Typicalselectable marker genes encode proteins that confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, or alternatively, are complement auxotrophicdeficiencies, or in other alternatives supply specific nutrients thatare not present in complex media, e.g., the gene encoding D-alanineracemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid, and hygromycin. Common selectablemarkers for mammalian cells are those that enable the identification ofcells competent to take up a nucleic acid encoding a humanizedanti-IL-36R antibody, such as DHFR (dihydrofolate reductase), thymidinekinase, metallothionein-I and -II (such as primate metallothioneingenes), adenosine deaminase, ornithine decarboxylase, and the like.Cells transformed with the DHFR selection gene are first identified byculturing all of the transformants in a culture medium that containsmethotrexate (Mtx), a competitive antagonist of DHFR. An appropriatehost cell when wild-type DHFR is employed is the Chinese hamster ovary(CHO) cell line deficient in DHFR activity (e.g., DG44).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding anti-IL-36R antibody, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH),can be selected by cell growth in medium containing a selection agentfor the selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See, e.g., U.S. Pat. No. 4,965,199.

Where the recombinant production is performed in a yeast cell as a hostcell, the TRP1 gene present in the yeast plasmid YRp7 (Stinchcomb etal., 1979, Nature 282: 39) can be used as a selectable marker. The TRP1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1(Jones, 1977, Genetics 85:12). The presence of the trp1 lesion in theyeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.Similarly, Leu2p-deficient yeast strains such as ATCC 20,622 and 38,626are complemented by known plasmids bearing the LEU2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis (Van den Berg, 1990, Bio/Technology8:135). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed (Fleer et al., 1991, Bio/Technology 9:968-975).

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid molecule encoding an anti-IL-36R antibody or polypeptide chainthereof. Promoters suitable for use with prokaryotic hosts include phoApromoter, β-lactamase and lactose promoter systems, alkalinephosphatase, tryptophan (trp) promoter system, and hybrid promoters suchas the tac promoter. Other known bacterial promoters are also suitable.Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding thehumanized anti-IL-36R antibody.

Many eukaryotic promoter sequences are known. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Inducible promoters have the additional advantage of transcriptioncontrolled by growth conditions. These include yeast promoter regionsfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,derivative enzymes associated with nitrogen metabolism, metallothionein,glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible formaltose and galactose utilization. Suitable vectors and promoters foruse in yeast expression are further described in EP 73,657. Yeastenhancers also are advantageously used with yeast promoters.

Humanized anti-IL-36R antibody transcription from vectors in mammalianhost cells is controlled, for example, by promoters obtained from thegenomes of viruses such as polyoma virus, fowlpox virus, adenovirus(such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, or from heat-shock promoters, providedsuch promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., 1982, Nature 297:598-601, disclosingexpression of human p-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous sarcoma virus long terminal repeat can be used as the promoter.

Another useful element that can be used in a recombinant expressionvector is an enhancer sequence, which is used to increase thetranscription of a DNA encoding a humanized anti-IL-36R antibody byhigher eukaryotes. Many enhancer sequences are now known from mammaliangenes (e.g., globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, an enhancer from a eukaryotic cell virus is used.Examples include the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers. See also Yaniv, 1982, Nature 297:17-18 for adescription of enhancing elements for activation of eukaryoticpromoters. The enhancer may be spliced into the vector at a position 5′or 3′ to the humanized anti-IL-36R antibody-encoding sequence, but ispreferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) can also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding anti-IL-36R antibody. One usefultranscription termination component is the bovine growth hormonepolyadenylation region. See WO94/11026 and the expression vectordisclosed therein. In some embodiments, humanized anti-IL-36R antibodiescan be expressed using the CHEF system. (See, e.g., U.S. Pat. No.5,888,809; the disclosure of which is incorporated by reference herein.)

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for humanizedanti-IL-36Rantibody-encoding vectors. Saccharomyces cerevisiae, orcommon baker's yeast, is the most commonly used among lower eukaryotichost microorganisms. However, a number of other genera, species, andstrains are commonly available and useful herein, such asSchizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis,K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii(ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906),K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichiapastors (EP 183,070); Candida; Trichoderma reesia (EP 244,234);Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis;and filamentous fungi such as, e.g., Neurospora, Penicillium,Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated humanizedanti-IL-36R antibody are derived from multicellular organisms. Examplesof invertebrate cells include plant and insect cells, including, e.g.,numerous baculoviral strains and variants and corresponding permissiveinsect host cells from hosts such as Spodoptera frugiperda(caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito),Drosophila melanogaster (fruitfly), and Bombyx mori (silk worm). Avariety of viral strains for transfection are publicly available, e.g.,the L-1 variant of Autographa californica NPV and the Bm-5 strain ofBombyx mori NPV, and such viruses may be used, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can also be utilized as hosts.

In another aspect, expression of humanized anti-IL-36R is carried out invertebrate cells. The propagation of vertebrate cells in culture (tissueculture) has become routine procedure and techniques are widelyavailable. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651), humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, (Graham et al., 1977, J. Gen Virol. 36: 59), babyhamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovarycells/-DHFR1 (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216; e.g., DG44), mouse sertoli cells (TM4, Mather, 1980, Biol. Reprod.23:243-251), monkey kidney cells (CV1 ATCC CCL 70), African green monkeykidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells(HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo ratliver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT060562, ATCC CCL51), TR1 cells (Mather et al., 1982, Annals N.Y. Acad.Sci. 383: 44-68), MRC 5 cells, FS4 cells, and human hepatoma line (HepG2).

Host cells are transformed with the above-described expression orcloning vectors for humanized anti-IL-36R antibody production andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

The host cells used to produce a humanized anti-IL-36R antibodydescribed herein may be cultured in a variety of media. Commerciallyavailable media such as Ham's F10 (Sigma-Aldrich Co., St. Louis, Mo.),Minimal Essential Medium ((MEM), (Sigma-Aldrich Co.), RPMI-1640(Sigma-Aldrich Co.), and Dulbecco's Modified Eagle's Medium ((DMEM),Sigma-Aldrich Co.) are suitable for culturing the host cells. Inaddition, any of the media described in one or more of Ham et al., 1979,Meth. Enz. 58: 44, Barnes et al., 1980, Anal. Biochem. 102: 255, U.S.Pat. No. 4,767,704, U.S. Pat. No. 4,657,866, U.S. Pat. No. 4,927,762,U.S. Pat. No. 4,560,655, U.S. Pat. No. 5,122,469, WO 90/103430, and WO87/00195 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as gentamicin), trace elements (defined as inorganiccompounds usually present at final concentrations in the micromolarrange), and glucose or an equivalent energy source. Other supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, the cells may bedisrupted to release protein as a first step. Particulate debris, eitherhost cells or lysed fragments, can be removed, for example, bycentrifugation or ultrafiltration. Carter et al., 1992, Bio/Technology10:163-167 describes a procedure for isolating antibodies that aresecreted to the periplasmic space of E. coli. Briefly, cell paste isthawed in the presence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 minutes. Cell debriscan be removed by centrifugation. Where the antibody is secreted intothe medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants. A variety of methodscan be used to isolate the antibody from the host cell.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing a typical purification technique. The suitability of protein A asan affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human gamma1, gamma2, orgamma4 heavy chains (see, e.g., Lindmark et al., 1983 J. Immunol. Meth.62:1-13). Protein G is recommended for all mouse isotypes and for humangamma3 (see, e.g., Guss et al., 1986 EMBO J. 5:1567-1575). A matrix towhich an affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a C_(H3) domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, reverse phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE™ chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, typically performed at low salt concentrations(e.g., from about 0-0.25M salt).

Also included are nucleic acids that hybridize under low, moderate, andhigh stringency conditions, as defined herein, to all or a portion(e.g., the portion encoding the variable region) of the nucleotidesequence represented by isolated polynucleotide sequence(s) that encodean antibody or antibody fragment of the present invention. Thehybridizing portion of the hybridizing nucleic acid is typically atleast 15 (e.g., 20, 25, 30 or 50) nucleotides in length. The hybridizingportion of the hybridizing nucleic acid is at least 80%, e.g., at least90%, at least 95%, or at least 98%, identical to the sequence of aportion or all of a nucleic acid encoding an anti-IL-36R polypeptide(e.g., a heavy chain or light chain variable region), or its complement.Hybridizing nucleic acids of the type described herein can be used, forexample, as a cloning probe, a primer, e.g., a PCR primer, or adiagnostic probe.

Non-Therapeutic Uses

The antibodies described herein are useful as affinity purificationagents. In this process, the antibodies are immobilized on a solid phasesuch a Protein A resin, using methods well known in the art. Theimmobilized antibody is contacted with a sample containing the IL-36Rprotein (or fragment thereof) to be purified, and thereafter the supportis washed with a suitable solvent that will remove substantially all thematerial in the sample except the IL-36R protein, which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent that will release the IL-36R protein from the antibody.

Anti-IL-36R antibodies, for example humanized anti-IL-36R antibodies,are also useful in diagnostic assays to detect and/or quantify IL-36Rprotein, for example, detecting IL-36R expression in specific cells,tissues, or serum. The anti-IL-36R antibodies can be used diagnosticallyto, for example, monitor the development or progression of a disease aspart of a clinical testing procedure to, e.g., determine the efficacy ofa given treatment and/or prevention regimen. Detection can befacilitated by coupling the anti-IL-36R antibody. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, radioactivematerials, positron emitting metals using various positron emissiontomographies, and nonradioactive paramagnetic metal ions. See, forexample, U.S. Pat. No. 4,741,900 for metal ions which can be conjugatedto antibodies for use as diagnostics according to the present invention.

The anti-IL-36R antibodies can be used in methods for diagnosing anIL-36R-associated disorder (e.g., a disorder characterized by abnormalexpression of IL-36R) or to determine if a subject has an increased riskof developing an IL-36R-associated disorder. Such methods includecontacting a biological sample from a subject with an IL-36R antibodyand detecting binding of the antibody to IL-36R. By “biological sample”is intended any biological sample obtained from an individual, cellline, tissue culture, or other source of cells potentially expressingIL-36R. Methods for obtaining tissue biopsies and body fluids frommammals are well known in the art.

In some embodiments, the method can further comprise comparing the levelof IL-36R in a patient sample to a control sample (e.g., a subject thatdoes not have an IL-36R-associated disorder) to determine if the patienthas an IL-36R-associated disorder or is at risk of developing anIL-36R-associated disorder.

It will be advantageous in some embodiments, for example, for diagnosticpurposes to label the antibody with a detectable moiety. Numerousdetectable labels are available, including radioisotopes, fluorescentlabels, enzyme substrate labels and the like. The label may beindirectly conjugated with the antibody using various known techniques.For example, the antibody can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody can be conjugated with a small hapten (such asdigoxin) and one of the different types of labels mentioned above isconjugated with an anti-hapten antibody (e.g., anti-digoxin antibody).Thus, indirect conjugation of the label with the antibody can beachieved.

Exemplary radioisotopes labels include ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. Theantibody can be labeled with the radioisotope, using the techniquesdescribed in, for example, Current Protocols in Immunology, Volumes 1and 2, 1991, Coligen et al., Ed. Wiley-Interscience, New York, N.Y.,Pubs. Radioactivity can be measured, for example, by scintillationcounting.

Exemplary fluorescent labels include labels derived from rare earthchelates (europium chelates) or fluorescein and its derivatives,rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin, andTexas Red are available. The fluorescent labels can be conjugated to theantibody via known techniques, such as those disclosed in CurrentProtocols in Immunology, for example. Fluorescence can be quantifiedusing a fluorimeter.

There are various well-characterized enzyme-substrate labels known inthe art (see, e.g., U.S. Pat. No. 4,275,149 for a review). The enzymegenerally catalyzes a chemical alteration of the chromogenic substratethat can be measured using various techniques. For example, alterationmay be a color change in a substrate that can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light that can be measured, using achemiluminometer, for example, or donates energy to a fluorescentacceptor.

Examples of enzymatic labels include luciferases such as fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (such asglucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocydic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described, for example, inO'Sullivan et al., 1981, Methods for the Preparation of Enzyme-AntibodyConjugates for use in Enzyme Immunoassay, in Methods in Enzym. (J.Langone & H. Van Vunakis, eds.), Academic press, N.Y., 73: 147-166.

Examples of enzyme-substrate combinations include, for example:Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate,wherein the hydrogen peroxidase oxidizes a dye precursor such asorthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB); alkaline phosphatase (AP) with para-Nitrophenylphosphate as chromogenic substrate; and β-D-galactosidase (β-D-Gal) witha chromogenic substrate such as p-nitrophenyl-β-D-galactosidase orfluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. No.4,275,149 and U.S. Pat. No. 4,318,980.

In another embodiment, the humanized anti-IL-36R antibody is usedunlabeled and detected with a labeled antibody that binds the humanizedanti-IL-36R antibody.

The antibodies described herein may be employed in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, and immunoprecipitation assays. See, e.g., Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

The anti-IL-36R antibody or antigen binding fragment thereof can be usedto inhibit the binding of ligand to the IL-36 receptor. Such methodscomprise administering an anti-IL-36R antibody or antigen bindingfragment thereof to a cell (e.g., a mammalian cell) or cellularenvironment, whereby signaling mediated by the IL-36 receptor isinhibited. These methods can be performed in vitro or in vivo. By“cellular environment” is intended the tissue, medium, or extracellularmatrix surrounding a cell. The anti-IL-36R antibody or antigen bindingfragment thereof is administered to the cellular environment of a cellin such a manner that the antibody or fragment is capable of binding toIL-36R molecules outside of and surrounding the cell, therefore,preventing the binding of IL-36 ligand to its receptor.

Diagnostic Kits

An anti-IL-36R antibody can be used in a diagnostic kit, i.e., apackaged combination of reagents in predetermined amounts withinstructions for performing the diagnostic assay. Where the antibody islabeled with an enzyme, the kit may include substrates and cofactorsrequired by the enzyme such as a substrate precursor that provides thedetectable chromophore or fluorophore. In addition, other additives maybe included such as stabilizers, buffers (for example a block buffer orlysis buffer), and the like. The relative amounts of the variousreagents may be varied widely to provide for concentrations in solutionof the reagents that substantially optimize the sensitivity of theassay. The reagents may be provided as dry powders, usually lyophilized,including excipients that on dissolution will provide a reagent solutionhaving the appropriate concentration.

Therapeutic Uses

In another embodiment, a humanized anti-IL-36R antibody disclosed hereinis useful in the treatment of various disorders associated with theexpression of IL-36R as described herein. Methods for treating an IL-36Rassociated disorder comprise administering a therapeutically effectiveamount of a humanized anti-IL-36R antibody to a subject in need thereof.

The humanized anti-IL-36R antibody or agent is administered by anysuitable means, including parenteral, subcutaneous, intraperitoneal,intrapulmonary, and intranasal, and, if desired for localimmunosuppressive treatment, intralesional administration (includingperfusing or otherwise contacting the graft with the antibody beforetransplantation). The humanized anti-IL-36R antibody or agent can beadministered, for example, as an infusion or as a bolus. Parenteralinfusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. In addition, thehumanized anti-IL-36R antibody is suitably administered by pulseinfusion, particularly with declining doses of the antibody. In oneaspect, the dosing is given by injections, most preferably intravenousor subcutaneous injections, depending in part on whether theadministration is brief or chronic.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on a variety of factors such as the type of diseaseto be treated, as defined above, the severity and course of the disease,whether the antibody is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the antibody, and the discretion of the attending physician. Theantibody is suitably administered to the patient at one time or over aseries of treatments.

Depending on the type and severity of the disease, about 1 μg/kg to 20mg/kg (e.g., 0.1-15 mg/kg) of antibody is an initial candidate dosagefor administration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment is sustaineduntil a desired suppression of disease symptoms occurs. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays. An exemplary dosingregimen is that disclosed in WO 94/04188.

The term “suppression” is used herein in the same context as“amelioration” and “alleviation” to mean a lessening of one or morecharacteristics of the disease.

The antibody composition will be formulated, dosed, and administered ina fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the antibody to be administeredwill be governed by such considerations, and is the minimum amountnecessary to prevent, ameliorate, or treat the disorder associated withIL-36R expression.

The antibody need not be, but is optionally, formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of humanizedanti-IL-36R23p19 antibody present in the formulation, the type ofdisorder or treatment, and other factors discussed above. These aregenerally used in the same dosages and with administration routes asused hereinbefore or about from 1 to 99% of the heretofore employeddosages.

Pharmaceutical Compositions and Administration Thereof

A composition comprising an IL-36R binding agent (e.g., an anti-IL-36Rantibody) can be administered to a subject having or at risk of havingan immunological disorder, respiratory disorder or a cancer. Theinvention further provides for the use of a IL-36R binding agent (e.g.,an anti-IL-36R antibody) in the manufacture of a medicament forprevention or treatment of a cancer, respiratory disorder orimmunological disorder. The term “subject” as used herein means anymammalian patient to which an IL-36Rbinding agent can be administered,including, e.g., humans and non-human mammals, such as primates,rodents, and dogs. Subjects specifically intended for treatment usingthe methods described herein include humans. The antibodies or agentscan be administered either alone or in combination with othercompositions in the prevention or treatment of the immunologicaldisorder, respiratory disorder or cancer. Such compositions which can beadministered in combination with the antibodies or agents includemethotrexate (MTX) and immunomodulators, e.g. antibodies or smallmolecules.

Examples of antibodies for use in such pharmaceutical compositions arethose that comprise a antibody or antibody fragment having the lightchain variable region amino acid sequence of any of SEQ ID NO: 1-10.Examples of antibodies for use in such pharmaceutical compositions arealso those that comprise a humanized antibody or antibody fragmenthaving the heavy chain variable region amino acid sequence of any of SEQID NO: 11-20.

Further examples of antibodies for use in such pharmaceuticalcompositions are also those that comprise a humanized antibody orantibody fragment having the light chain variable region amino acidsequence of any of SEQ ID NO:76-86. Preferred antibodies for use in suchpharmaceutical compositions are also those that comprise a humanizedantibody or antibody fragment having the heavy chain variable regionamino acid sequence of any of SEQ ID NO:87-101.

Further examples of antibodies for use in such pharmaceuticalcompositions are also those that comprise a humanized antibody orantibody fragment having the light chain variable region and heavy chainvariable region of any of SEQ ID NO: 77 and 89, SEQ ID NO: 80 and 88,SEQ ID NO: 80 and 89, SEQ ID NO: 77 and 87, SEQ ID NO: 77 and 88, SEQ IDNO: 80 and 87, SEQ ID NO: 86 and 100, SEQ ID NO: 85 and 101, or SEQ IDNO: 85 and 10.

Further examples of antibodies for use in such pharmaceuticalcompositions are also those that comprise a humanized antibody havingthe light chain region amino acid sequence of any of SEQ ID NO:115, 118,123 or 124. Preferred antibodies for use in such pharmaceuticalcompositions are also those that comprise humanized antibody having theheavy chain variable region amino acid sequence of any of SEQ ID NO:125,126, 127, 138 or 139.

Further examples of antibodies for use in such pharmaceuticalcompositions are also those that comprise Antibody B1, Antibody B2,Antibody B3, Antibody B4, Antibody B5, Antibody B6, Antibody C1,Antibody C2 or Antibody C3.

Various delivery systems are known and can be used to administer theIL-36R binding agent. Methods of introduction include but are notlimited to intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, and oral routes. The IL-36R bindingagent can be administered, for example by infusion, bolus or injection,and can be administered together with other biologically active agentssuch as chemotherapeutic agents. Administration can be systemic orlocal. In preferred embodiments, the administration is by subcutaneousinjection. Formulations for such injections may be prepared in forexample prefilled syringes that may be administered once every otherweek.

In specific embodiments, the IL-36R binding agent composition isadministered by injection, by means of a catheter, by means of asuppository, or by means of an implant, the implant being of a porous,non-porous, or gelatinous material, including a membrane, such as asialastic membrane, or a fiber. Typically, when administering thecomposition, materials to which the anti-IL-36R antibody or agent doesnot absorb are used.

In other embodiments, the anti-IL-36R antibody or agent is delivered ina controlled release system. In one embodiment, a pump may be used (see,e.g., Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref.Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek etal., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymericmaterials can be used. (See, e.g., Medical Applications of ControlledRelease (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974);Controlled Drug Bioavailability, Drug Product Design and Performance(Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983,Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985,Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard etal., 1989, J. Neurosurg. 71:105.) Other controlled release systems arediscussed, for example, in Langer, supra.

An IL-36R binding agent (e.g., an anti-IL-36R antibody) can beadministered as pharmaceutical compositions comprising a therapeuticallyeffective amount of the binding agent and one or more pharmaceuticallycompatible ingredients.

In typical embodiments, the pharmaceutical composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous or subcutaneous administration to human beings.Typically, compositions for administration by injection are solutions insterile isotonic aqueous buffer. Where necessary, the pharmaceutical canalso include a solubilizing agent and a local anesthetic such aslignocaine to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where thepharmaceutical is to be administered by infusion, it can be dispensedwith an infusion bottle containing sterile pharmaceutical grade water orsaline. Where the pharmaceutical is administered by injection, anampoule of sterile water for injection or saline can be provided so thatthe ingredients can be mixed prior to administration.

Further, the pharmaceutical composition can be provided as apharmaceutical kit comprising (a) a container containing a IL-36Rbinding agent (e.g., an anti-IL-36R antibody) in lyophilized form and(b) a second container containing a pharmaceutically acceptable diluent(e.g., sterile water) for injection. The pharmaceutically acceptablediluent can be used for reconstitution or dilution of the lyophilizedanti-IL-36R antibody or agent. Optionally associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

The amount of the IL-36R binding agent (e.g., anti-IL-36R antibody) thatis effective in the treatment or prevention of an immunological disorderor cancer can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and thestage of immunological disorder or cancer, and should be decidedaccording to the judgment of the practitioner and each patient'scircumstances. Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

Generally, the dosage of an anti-IL-36R antibody or IL-36R binding agentadministered to a patient with an immunological disorder orIL-36R-expressing cancer is typically about 0.1 mg/kg to about 100 mg/kgof the subject's body weight. The dosage administered to a subject isabout 0.1 mg/kg to about 50 mg/kg, about 1 mg/kg to about 30 mg/kg,about 1 mg/kg to about 20 mg/kg, about 1 mg/kg to about 15 mg/kg, orabout 1 mg/kg to about 10 mg/kg of the subject's body weight.

Exemplary doses include, but are not limited to, from 1 ng/kg to 100mg/kg. In some embodiments, a dose is about 0.5 mg/kg, about 1 mg/kg,about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg,about 11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15mg/kg or about 16 mg/kg. The dose can be administered, for example,daily, once per week (weekly), twice per week, thrice per week, fourtimes per week, five times per week, six times per week, biweekly ormonthly, every two months, or every three months. In specificembodiments, the dose is about 0.5 mg/kg/week, about 1 mg/kg/week, about2 mg/kg/week, about 3 mg/kg/week, about 4 mg/kg/week, about 5mg/kg/week, about 6 mg/kg/week, about 7 mg/kg/week, about 8 mg/kg/week,about 9 mg/kg/week, about 10 mg/kg/week, about 11 mg/kg/week, about 12mg/kg/week, about 13 mg/kg/week, about 14 mg/kg/week, about 15mg/kg/week or about 16 mg/kg/week. In some embodiments, the dose rangesfrom about 1 mg/kg/week to about 15 mg/kg/week.

In some embodiments, the pharmaceutical compositions comprising theIL-36R binding agent can further comprise a therapeutic agent, eitherconjugated or unconjugated to the binding agent. The anti-IL-36Rantibody or IL-36R binding agent can be co-administered in combinationwith one or more therapeutic agents for the treatment or prevention ofimmunological disorders or cancers.

Such combination therapy administration can have an additive orsynergistic effect on disease parameters (e.g., severity of a symptom,the number of symptoms, or frequency of relapse).

With respect to therapeutic regimens for combinatorial administration,in a specific embodiment, an anti-IL-36R antibody or IL-36R bindingagent is administered concurrently with a therapeutic agent. In anotherspecific embodiment, the therapeutic agent is administered prior orsubsequent to administration of the anti-IL-36R antibody or IL-36Rbinding agent, by at least an hour and up to several months, for exampleat least an hour, five hours, 12 hours, a day, a week, a month, or threemonths, prior or subsequent to administration of the anti-IL-36Rantibody or IL-36R binding agent.

Articles of Manufacture

In another aspect, an article of manufacture containing materials usefulfor the treatment of the disorders described above is included. Thearticle of manufacture comprises a container and a label. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. The containers may be formed from a variety of materials such asglass or plastic. The container holds a composition that is effectivefor treating the condition and may have a sterile access port. Forexample, the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle. The activeagent in the composition is the humanized anti-IL-36R antibody. Thelabel on or associated with the container indicates that the compositionis used for treating the condition of choice. The article of manufacturemay further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution, and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

The invention is further described in the following examples, which arenot intended to limit the scope of the invention.

The antibodies of the present invention are further described in theExamples below.

EXAMPLES Example 1 Identification of Anti-Human IL-36R Antibodies

Multiple mouse strains were immunized with recombinantly produced humanIL-36R (ECD—extracellular domain: amino acids 20-332 of GenbankAccession #NP_003845) protein and those which generated a strong titerresponse taken into traditional hybridoma generation. Fusion productseliciting a strong binding to human IL-36R (ECD) yet no binding to humanIL-1R1 (the most related IL-1R family member) were subcloned andre-screened. Multiple hybridomas were identified to yield monoclonalantibodies which bound and neutralized signaling from IL-36R (seeexamples 2, 3, and 4). Variable domains were cloned from the hybridomasusing standard PCR primer sets. The variable domains and specific CDRsof representative monoclonal antibodies are described above. All of themouse antibodies were converted to chimeric antibodies consisting of themouse variable domains on human constant domains (hu IgG1 KO/kappa). Thehu IgG1 KO (knock out) has two replacement mutations (Leu234Ala andLeu235Ala) that eliminate ADCC and CDC activity by reducing effectorfunctions such as FcγR and complement binding. The variable domains ofthe mouse and chimeric antibodies are identical. Chimeric antibodies aregenerated to confirm the function of the antibody and to ensure thecorrect variable domain sequence has been obtained.

Example 2 Molecular Binding Affinities of Identified Mouse Anti-humanIL-36R Antibodies

A) Kinetics and binding affinities of anti-IL-36R antibodies binding torecombinant human IL-36R were measured using the Proteon (Bio-Rad,Hercules, Calif.) using material generated from hybridoma followingsingle column purification. The binding affinities of all the mouseleads to human IL-36R run at a single IL-36R surface coat concentrationwas estimated to be <100 pM. The binding affinities of the mouseantibodies to human IL-36R run at 7 different surface densities(globally fit) is shown in Table 1. Binding of the chimeric anti-IL36RIgGs is equivalent to the respective mouse leads.

TABLE 1 Binding affinity of mouse anti-human IL-36R antibodies. LeadBinding K_(D) (pM) Mouse 73C5 57 Mouse 73F6 25 Mouse 78C8 63 Mouse 81A116 Mouse 81B4 24 Mouse 33D10  9

B) Molecular selectivity over mouse IL-36R and human IL-1R1 The mouseand chimeric anti-IL-36R antibodies were also injected over either amouse IL-36R or a human IL-1R1 surface at a concentration of 100 nM. Thebinding signal to mouse IL-36R and to human IL-1R1 for these antibodiesmeasured using the Fortebio Octet (Fortebio, Menlo Park, Calif.) iszero, which indicates these antibodies selectively bind to human IL-36R.The binding of the anti-IL-36R antibodies to human IL-36R was alsoanalyzed in the presence of 50% human serum and no significant effect ofserum on binding on-rate was observed demonstrating high specificity.

Example 3 Potency of Mouse and Chimeric Anti-Human IL-36R Antibodies inFunctional Human and Cynomolgus Assays

Protocols: Human NCI/ADR-RES Cells pNFκB/Cytokine Release Assays

Reagents:

R&D Systems: truncated rh IL36βR&D Systems: truncated rh IL36γR&D Systems: truncated rh IL36α:

MA6000 Phospho-NFκB (Ser536) Whole Cell Lysate Kit Meso ScaleDiagnostics, LLC

MSD ELISA custom human 96-well 4-spot assay

Meso Scale Diagnostics, LLC

NCI/ADR-RES cells were plated at 45000 cells/well, in RPMI media with0.25% serum in a 96well plate. One plate was utilized for the analysisof pNFκB and another for cytokine release. The plates were thenincubated overnight at 37° C., 5% CO_(2.) Ligands (IL36α, β, or γ) andantibodies were diluted at 4× desired concentration in serum starved(SS) media. Antagonists (antibodies) were added to cells prior toligand. For pNFκB: NCI cells +/−ligand and antagonist were incubated for1 hour, 37° C., 5% CO_(2.) Media was then aspirated and cells were lysedin 100 μl/well Complete lysis Buffer on ice 30 min. Lysate was thencentrifuged at 2500 RPM, 20 min, 4° C., and transferred to an MSD ELISAplate and assayed for pNFκB as per the manufacturer's protocol. ForCytokine release: 18-24 hours after stimulation, supernatants weretransferred to an MSD ELISA plate and assayed for cytokine as permanufacturer's protocol.

Protocol: pNFκB (S536) MSD ELISA for BaF/3 Cynomolgus IL-36R Cells

BaF/3 cynomolgus IL-36R cells were plated at 90,000 cells/well in SSmedia in a 96well plate. 100 ul media was added to control wells.Antagonists (antibodies) were diluted at 4× desired concentration and 50μl was added to each well. Ligands (IL36α, β, or γ) were diluted at 4×desired concentration in SS media and 50 μl were added to each well (forfinal volume of 200 μl). Plates were incubated for 15 min, 37° C., 5%CO_(2.) Plates were centrifuged briefly, media was aspirated and cellswere lysed in 100 μl/well Complete Lysis Buffer (see MSD pNFκB protocol)and incubated on ice 30 min. Lysates were then centrifuged at 2500 RPM,20 min, 4° C. and transferred to an MSD ELISA plate. Lysates were thenevaluated for pNFκB activity using the MSD kit as described above.

Results: IC₉₀ results for mouse anti-human IL-36R antibodies in humanfunctional assays (pNFκB and cytokine release) and a cynomolgusfunctional assay (pNFkB) with human IL-36 ligands are shown in Table 2.IC₉₀ results for chimeric anti-human IL-36R antibodies in humanfunctional assays (pNFκB and cytokine release) and a cynomolgusfunctional assay (pNFκB) with human IL-36 ligands are shown in Table 3.

TABLE 2 Potency of mouse antibodies in functional cell assays (isotypecontrols demonstrated no inhibition of activity at highest concentrationtested for samples) NCI/ADR-RES 33D10 73C5 73F6F8 76E10E8 78C8D1 81A1D181B4E11 89A12B8 172C8B12 67E7E8 pNFκB trun-IL-36a 1.7 3.4 2.8 ND 5.8 2.71.7 ND ND ND IC90 (nM) trun-IL-36b 1.3 3.4 3 ND 6.8 4.2 1.4 ND ND NDIC90 (nM) trun-IL-36g 1.1 2.5 1.5 1.9 4.6 2.2 1.2 145 3.8 >67 IC90 (nM)GM-CSF trun-IL-36a 0.8 4.8 5.9 ND 7.1 1.2 0.8 ND ND ND IC90 (nM)trun-IL-36b 0.6 1.4 1.7 ND 2 1.2 0.3 ND ND ND IC90 (nM) trun-IL-36g 0.52 1.4 ND 1.7 0.7 0.4 ND ND ND IC90 (nM) IL-6 trun-IL-36a 0.8 19 19 ND 1417 0.8 ND ND ND IC90 (nM) trun-IL-36b 0.5 3.8 5.1 ND 5.8 5.3 0.3 ND NDND IC90 (nM) trun-IL-36g 0.3 1.9 2.2 ND 1.3 0.8 0.2 ND ND ND IC90 (nM)IL-8 trun-IL-36a 0.6 3.3 3.2 ND 4.7 1.9 0.7 ND ND ND IC90 (nM)trun-IL-36b 0.6 0.6 0.8 ND 1.1 0.5 0.2 ND ND ND IC90 (nM) trun-IL-36g0.5 1.4 0.9 ND 1.4 0.5 0.3 ND ND ND IC90 (nM) BaF cyno pNFκBtrun-IL-36a >4000 0.2 0.5 >4000 1 0.6 2.4 >4000 >4000 >4000 IC90 (nM)trun-IL-36b >333 1.6 0.7 >333 2.1 0.9 3.5 >333 >333 >333 IC90 (nM)trun-IL-36g >333 1.5 0.8 >333 1.5 1 5.1 >333 >333 >333 IC90 (nM)

TABLE 3 Potency of chimeric antibodies in functional cell assays(isotype controls demonstrated no inhibition of activity at highestconcentration tested for samples) NCI/ADR-RES C33D10 C73C5 C81B4 pNFκBtrun-IL-36a IC90 1.9 2.1 1.3 (nM) trun-IL-36b IC90 2 0.9 0.9 (nM)trun-IL-36g IC90 1.3 1.8 0.4 (nM) GM-CSF trun-IL-36a IC90 0.3 0.9 ND(nM) trun-IL-36b IC90 0.3 1.1 ND (nM) trun-IL-36g IC90 0.3 0.7 ND (nM)IL-6 trun-IL-36a IC90 0.4 2.1 ND (nM) trun-IL-36b IC90 0.6 6.7 ND (nM)trun-IL-36g IC90 0.7 6 ND (nM) IL-8 trun-IL-36a IC90 0.4 0.7 0.4 (nM)trun-IL-36b IC90 0.3 0.4 0.1 (nM) trun-IL-36g IC90 0.2 0.4 0.1 (nM) TNFαtrun-IL-36a IC90 2.4 2.5 ND (nM) trun-IL-36b IC90 0.9 7.9 ND (nM)trun-IL-36g IC90 0.6 1.3 ND (nM) BaF cyno — pNFκB trun-IL-36a IC90 ND1.2 42 (nM) trun-IL-36b IC90 ND 4.1 48 (nM) trun-IL-36g IC90 ND 4.3 28(nM)

Example 4 Binding of Mouse Anti-Human IL-36R Antibodies to Human IL-36RExpressing Cells Protocol for Binding of Antibodies by Flow Cytometry

HEK293 cells transfected with full-length human IL-36R or NCI/ADR-REScells were passaged for 24 hours prior to staining. Cells were removedfrom flasks by rinsing with 10 ml of 5 mM EDTA in PBS, and thenincubated at 37° C. for 10 min with an additional 10 ml of 5 mM EDTA and2.5 ml of Accumax to declump/disperse cells. Antibodies were thendiluted to specified concentrations in PBS+2% BSA, and cells incubatedfor 20 min at room temperature. Excess antibody was then washed byadding 200 μl of PBS and then centrifuged. Secondary reagent was thenadded at 50 μl per well and cells are incubated for 15 min at roomtemperature and then washed as above. Cells were resuspended in 200 μlPBS and analyzed by flow cytometry. The binding EC₅₀'s for the mouseanti-human IL-36R antibodies binding to human IL-36R HEK transfectantsare shown in Table 4.

TABLE 4 EC₅₀(M) binding to Clone HEK-IL-36R cells 33D10 4.748e−010 67E75.321e−010 73F6 7.456e−010 76E10 4.257e−010 78C8 5.289e−010 81A12.795e−010 81B4 3.016e−010 89A12 6.089e−010

Example 5 Production of Humanized IL-36R Antibodies

In order to reduce potential immunogenicity following administration inman the mouse anti-human IL-36R monoclonal antibodies 81B4 and 73C5 were‘humanized’ through a design and screening process. Human frameworksequences were selected for the mouse leads based on the frameworkhomology, CDR structure, conserved canonical residues, conservedinterface packing residues and other parameters. The specificsubstitution of amino acid residues in these framework positions canimprove various aspects of antibody performance including bindingaffinity and/or stability, over that demonstrated in humanizedantibodies formed by “direct swap” of CDRs or HVLs into the humangermline framework regions. Fabs that showed better or equal binding andimproved expression as compared to the chimeric parent Fab were selectedfor further characterization. Representative humanized variable regionsfor antibody 81B4 and 73C5 are shown in are shown the specificationsection. In this manner, Antibody B1 to Antibody B6 were humanizedantibodies derived from mouse antibody 81B4 (cloned into a human IgG1 KO(KO=knock-out)/kappa backbone. Antibodies B1 to B6 are shown in Table A.Antibody C1 to Antibody C3 were humanized antibodies derived from mouseantibody 73C5 (cloned into a human IgG1-KO (KO=knock-out)/kappabackbone. Antibodies C1 to C3 are shown in Table C.

Example 6 Binding of Humanized IL-36R Antibodies

Kinetics and binding affinities of humanized anti-IL-36R antibodiesbinding to recombinant human IL-36R were measured using the Proteon(Bio-Rad, Hercules, Calif.). Human IL-36R was immobilized at 5 differentsurface densities and results analyzed using global fit (see Table 5showing results of three experiments). Binding of the humanizedantibodies to NCI/ADR-RES cells via flow cytometry was measured usingprotocol described in Example 4 (See Table 5 for EC₉₀ values).

TABLE 5 Molecular and Cellular Binding affinities of humanizedanti-human IL-36R antibodies. K_(D) ± Standard Deviation Antibody (LC +HC) (pM) EC₉₀ (nM) B1 − 32_138 + 33_49 27 ± 2.5 2.0 B2 − 32_138 + 33_8532 ± 5.4 2.3 B3 − 32_138 + 33_90 20 ± 2.2 1.4 B4 − 32_105 + 33_85 35 ±3.7 1.5 B5 − 32_105 + 33_90 24 ± 7.6 1.7 B6 − 32_105 + 33_49 41 ± 4.91.7

Example 7 Potency of Humanized Anti-Human IL-36R Antibodies inFunctional Human Assays

Functional blockade of signaling with the humanized IL-36R variants fromhuman NCI/ADR-RES cells were tested as described in Example 3. IC₉₀results for the humanized anti-human IL-36R antibodies in humanfunctional assays (pNFκB and cytokine release) with human IL-36 ligandsare shown in Table 6.

TABLE 6 Potency [IC₉₀ (nM)] of humanized antibodies in human functionalNCI/ADR-RES cell assays (Results equal averages of at least 2experiments. Iso- type controls demonstrated no inhibition of activityat highest concentration tested for samples) Ligand B1 B2 B3 B4 B5 B6 C1C2 C3 pNFκB trun-IL-36a 3.9 5.3 2.3 1.7 2.6 1.8 4.6 9.7 9.8 trun-IL-36b3.4 3.4 2.9 2.2 2.8 2.4 5.5 8.6 5.6 trun-IL-36g 2.5 2.3 2.1 1.5 1.5 1.53.4 6.1 5.1 IL-8 trun-IL-36a 4.0 2.8 2.3 2.6 2.6 2.2 NA NA NAtrun-IL-36b 3.2 2.9 2.7 2.9 2.5 2.3 NA NA NA trun-IL-36g 4.2 3.5 3.4 3.22.8 2.4 NA NA NA

Example 8 Potency of Anti-IL-36R Antibodies in Functional Human PrimaryKeratinocyte Assays

Protocols: Human Primary Epidermal Keratinocyte pNFkB/Cytokine ReleaseAssays

Cells were plated at 30,000 cells/well in culture media in 96 wellplates and incubated overnight at 37° C., 5% CO_(2.) Assays were thenperformed as described in Example 3. Results: IC₉₀ results for mouse,chimeric, and humanized anti-IL-36R antibodies in human primarykeratinocyte assays (pNFkB and IL-8 release) stimulated with human IL-36ligands are shown in Table 7, Table 8, and Table 9, respectively.

TABLE 7 (isotype controls demonstrated no inhibition of activity athighest concentration tested for samples) NHK 33D10 73C5 73F6 78C8 81A181B4 pNFκB trun-IL-36a 1.8 10.7 8.2 11.6 ND 1.3 IC90 (nM) trun-IL-36b2.3 14.4 5.2 14.2 ND 1.1 IC90 (nM) trun-IL-36g 0.8 1.7 1.3 10.6 ND 0.5IC90 (nM) IL-8 trun-IL-36a 1.5 20.8 17.8 19.3 ND 0.7 IC90 (nM)trun-IL-36b 3.0 46 26 34 ND 1.2 IC90 (nM) trun-IL-36g 0.8 2.4 2.1 3.6 ND0.5 IC90 (nM)

TABLE 8 Potency of chimeric anti-IL-36R antibodies in primary humankeratinocyte assays (isotype controls demonstrated no inhibition ofactivity at highest concentration tested for samples) NHK C73C5 C81A1C81B4 pNFκB trun-IL-36a 20.5 ND 1.4 IC90 (nM) trun-IL-36b 8.3 7.1 1.8IC90 (nM) trun-IL-36g 1.4 ND 0.5 IC90 (nM) IL-8 trun-IL-36a 29.7 23.41.2 IC90 (nM) trun-IL-36b 49.3 87 11.1 IC90 (nM) trun-IL-36g 3.6 1.2 0.3IC90 (nM)

TABLE 9 Potency of humanized anti-IL-36R antibodies in primary humankeratinocyte assays (isotype controls demonstrated no inhibition ofactivity at highest concentration tested for samples) NHK BI 1 BI 2 BI 3pNFκB trun-IL-36a 2.9 2.1 2.2 IC90 (nM) trun-IL-36b 3.6 2.9 1.9 IC90(nM) trun-IL-36g 1.0 1.3 0.7 IC90 (nM) IL-8 trun-IL-36a 2.3 2.8 1.8 IC90(nM) trun-IL-36b 3.9 4.1 3.5 IC90 (nM) trun-IL-36g 0.8 0.8 0.8 IC90 (nM)

Example 9 Potency of Anti-IL-36R Antibodies in Functional Human PrimaryIntestinal Epithelial Cell Assays

Protocols: Human Primary Intestinal Epithelial Cell pNFkB/CytokineRelease Assays

Cells were plated at 30,000 cells/well in culture media in 96well plateand incubated overnight at 37° C., 5% CO_(2.) Assays were then performedas described in Example 3.

Results: IC₉₀ results for mouse anti-IL-36R antibodies in human primaryintestinal epithelial cell assays (pNFkB and IL-8 release) stimulatedwith human IL-36 ligands are shown in Table 10.

TABLE 10 Potency of mouse anti-IL-36R antibodies in primary humanintestinal epithelial cell assays (isotype controls demonstrated noinhibition of activity at highest concentration tested for samples) HIE73C5 81B4 pNFκB trun-IL-36a IC90 21 1.3 (nM) trun-IL-36b IC90 20 7.4(nM) trun-IL-36g IC90 32 9.5 (nM) IL-8 trun-IL-36a IC90 123 5.8 (nM)trun-IL-36b IC90 154 9.8 (nM) trun-IL-36g IC90 ND 16 (nM)

Example 10 Potency of Anti-IL-36R Antibodies in Functional Human PrimaryIntestinal Myofibroblast Assays

Protocols: Human Primary Intestinal Myofibroblast pNFκB/Cytokine ReleaseAssays

Cells were plated at 30,000 cells/well in culture media in 96well plateand incubated at 37° C., 5% CO_(2.) Assays were then performed asdescribed in Example 3.

Results: IC₉₀ results for anti-IL-36R antibodies in human primaryintestinal myofibroblast assays (pNFkB and IL-8 release) stimulated withhuman IL-36 ligands are shown in Table 11 and Table 12.

TABLE 11 Potency of mouse and chimeric anti-IL-36R antibodies in primaryhuman intestinal myofibroblast assays (isotype controls demonstrated noinhibition of activity at highest concentration tested for samples) HIM81B4 C81B4 pNFκB trun-IL-36a IC90 7 6.2 (nM) trun-IL-36b IC90 4 2.3 (nM)trun-IL-36g IC90 3.6 0.9 (nM) IL-8 trun-IL-36a IC90 2 2.3 (nM)trun-IL-36b IC90 2.3 1.4 (nM) trun-IL-36g IC90 2.9 2 (nM)

TABLE 12 Potency of humanized anti-IL-36R antibodies in primary humanintestinal myofibroblast assays (isotype controls demonstrated noinhibition of activity at highest concentration tested for samples) HIMB3 B5 B6 pNFkB trun-IL-36a 9.8 5.4 3.2 IC90 (nM) trun-IL-36b 3.7 1.7 2.8IC90 (nM) trun-IL-36g 2.6 1.7 3.7 IC90 (nM) IL-8 trun-IL-36a 4.4 3.8 2.1IC90 (nM) trun-IL-36b 2.8 2.6 2.0 IC90 (nM) trun-IL-36g 5.8 6.7 5.2 IC90(nM)

Example 11 Potency of Anti-IL-36R Antibodies in Functional Human PrimaryDermal Fibroblast Assays

Protocols: Human Primary Dermal Fibroblast pNFκB/Cytokine Release Assays

Cells were plated at 30,000 cells/well in culture media in 96well plateand incubated overnight at 37° C., 5% CO_(2.) Assays were then performedas described in Example 3.

Results: IC₉₀ results for anti-IL-36R antibodies in human primary dermalfibroblast assays (pNFkB and IL-8 release) stimulated with human IL-36ligands are shown in Table 13 and Table 14.

TABLE 13 Potency of mouse and chimeric anti-IL-36R antibodies in primaryhuman dermal fibroblast assays (isotype controls demonstrated noinhibition of activity at highest concentration tested for samples) HDF81B4 C81B4 pNFκB trun-IL-36a IC90 1.9 1.8 (nM) trun-IL-36b IC90 4.9 3.4(nM) trun-IL-36g IC90 3.9 7.4 (nM) IL-8 trun-IL-36a IC90 0.9 0.6 (nM)trun-IL-36b IC90 0.4 0.5 (nM) trun-IL-36g IC90 0.4 0.3 (nM)

TABLE 14 Potency of humanized anti-IL-36R antibodies in primary humandermal fibroblast assays (isotype controls demonstrated no inhibition ofactivity at highest concentration tested for samples) HDF B3 B5 B6 pNFκBtrun-IL-36a IC90 6.4 10.2 3.8 (nM) trun-IL-36b IC90 13.5 9.9 9 (nM)trun-IL-36g IC90 10 8.6 16.8 (nM) IL-8 trun-IL-36a IC90 2.1 1.5 1.4 (nM)trun-IL-36b IC90 1.3 1.2 1 (nM) trun-IL-36g IC90 1.1 1.1 0.7 (nM)

Example 12 Potency of Mouse Anti-IL-36R Antibodies in Functional HumanPrimary Proximal Tubular Cell Assays

Protocol: Human Primary Proximal Tubular Cells pNFkB/Cytokine ReleaseAssays.

Cells were plated at 5,000 cells/well in culture media in 96well plateand incubated overnight at 37° C., 5% CO_(2.) Assays were performed asdescribed in Example 3.

Results: IC₉₀ results for mouse, chimeric, and humanized anti-IL-36Rantibodies in human primary proximal tubular cell assays (IL-8 release)stimulated with human IL-36 ligands are shown in Table 15.

TABLE 15 Potency of mouse and human anti-IL-36R antibodies in primaryhuman proximal tubular cell assays (isotype controls demonstrated noinhibition of activity at highest concentration tested for samples) HPT81B4 B3 B5 B6 IL-8 trun-IL-36a .04 ND 5 ND IC90 (nM) trun-IL-36b .01 ND5 ND IC90 (nM) trun-IL-36g .04 ND 3 ND IC90 (nM)

Example 13 Inhibition of IL-8 Production from IL-36γ StimulatedReconstructed Human Epidermis Protocol Reconstructed Epidermis

Anti-IL-36R antibodies (1.5 μg/ml) were pre-incubated with reconstructedhuman epidermis and stimulated with human recombinant IL-36γ (20 ng/ml).Recombinant human IL-1β (20 ng/ml; R & D Systems) was used as a positivecontrol. After 24 hours in culture, cell supernatants were collected andassayed for IL-8 (assays for IL-8 are described in Example 3). Sampleswere tested in triplicate and the average pg/ml±standard error is shownin the table below (Table 16).

TABLE 16 Average IL-8 (pg/ml) Antibody Cytokine Stimulation +/− StandardError No antibody None  57.3 ± 15.3 33D10 None 15.8 ± 0.7 No antibody 20ng/mL IL-1β 158.9 ± 13.3 33D10 20 ng/mL IL-1β 168.5 ± 22.6 No antibody20 ng/mL IL-36y 142.1 ± 22.2 33D10 20 ng/mL IL-36y 38.63 ± 6.7 

Example 14 Inhibition of IL-36 Ligand Induced S100A7 and S100A12 GeneExpression in Reconstructed Human Epidermis

Stimulation of reconstructed human epidermis with agonsitic IL-36ligands induces S100A7 and S100A12 gene expression. S100A7 and S100A12are genes located within the epidermal differentiation complex.

Protocol:

Reconstructed human epidermis were incubated with anti-IL-36R antibodies(1.5 μg/ml) and stimulated with human recombinant IL-36γ (20 ng/ml).Recombinant human IL-1β (20 ng/mL; R & D Systems) was used as a positivecontrol. After 24 hours in culture at 5% CO₂ and 37° C., RNA wasisolated from the reconstructed human epidermis and assayed for geneexpression by real-time reverse trancriptase-polymerase chain reaction.Relative expression was calculated using the 2^(−ΔΔct) method. Sampleswere tested in triplicate and the average expression±standard error isshown in the table below (Table 17).

TABLE 17 Mean S100A7 Mean S100A12 Cytokine Expression +/− Expression +/−Antibody Stimulation Standard Error Standard Error No antibody None 1.00± 0.79 1.00 ± 0.47 33D10 None 3.92 ± 0.36 1.93 ± 0.02 No antibody 20ng/mL IL-1β 76.03 ± 24.66 47.84 ± 9.24  33D10 20 ng/mL IL-1β 95.83 ±11.83 76.41 ± 6.92  No antibody 20 ng/mL IL-36y 19.57 ± 3.26  20.53 ±5.21  33D10 20 ng/mL IL-36y 3.47 ± 1.37 2.01 ± 0.35

Example 15 Efficacy of Anti-IL-36R Antibody in Xenotransplant Model ofPsoriasis Protocol:

Blood and non-lesional skin biopsies were obtained from 24 psoriasispatients that were clinically diagnosed by a dermatologist. Skinbiopsies were transplanted onto immune-deficient NIH-Ill mice andallowed to engraft for a period of four to five weeks.

Peripheral blood mononuclear cells (PBMC) were isolated from bloodcollected from each donor at the time of biopsy for intradermalinjection into the engrafted skin. Prior to injection, PBMC werestimulated with with 1 μg/ml Staphylococcal Enterotoxin B (ToxinTechnologies, Florida, USA) and 80 U/ml human recombinant IL-2(Peprotech Inc., Oosterhout, The Netherlands). Autologous PBMC wereintradermally injected with 7.5×10{circle around ( )}5 cells in PBS tosynchronize the induction of skin inflammation and the psoriasisphenotype. Three weeks after the injection of cells, the biopsies wereretrieved from the mice and analyzed by histology.

Histological staining was performed on cryo-preserved skin tissue of allgroups. Diagonal cross sections (8 μm), covering all skin-layers, wereprepared as described in FIG. 4. For assessment of epidermal thickness,two non-serial sections were randomly chosen from the center of thebiopsy and stained with haematoxylin-eosin. Subsequently, sections wereevaluated at a 100-fold magnification. Over the entire length of thebiopsy, ridge lengths were measured in both sections using an OlympusDP71 camera and Cell{circle around ( )}D imaging software (V2.7,Munster, Germany). Ridge length is defined as: the distance between theupper edge of the stratum granulosum to the bottom of the ridge.Biopsies were scored at random and in a blinded fashion.

Results for the average epidermal thickness and maximum epidermalthickness for each treatment group are shown in Table 18. Results forthe net change in epidermal thickness in each treatment group are shownin Table 19.

TABLE 18 Average Epidermal Maximum Epidermal Thickness (μM) Thickness(μM) Treatment mean SD N SEM mean SD N SEM Untreated 101.4 34.4 16 8.6147.1 35.0 16 8.7 Vehicle 106.0 31.9 9 10.6 151.6 48.9 9 16.3 33D10 93.424.1 10 7.6 130.9 30.8 10 9.7

TABLE 19 Average Epidermal Maximum Epidermal Thickness (μM) Thickness(μM) Treatment mean SD N SEM mean SD N SEM Untreated 36.2 31.9 32 5.652.6 40.0 32 7.1 Vehicle 43.5 30.9 18 7.3 63.4 52.0 18 12.2 33D10 27.826.6 20 5.9 35.1 34.1 20 7.6

Example 16 The Sub-Chronic Pulmonary Inflammation after 3 Weeks ofCigarette Smoke Exposure in Wild Type and Interleukin-1Receptor-Like 2Homozygous Knockout Mice Protocol:

Wild type or interleukin-1 receptor-like 2 mice were exposed tocigarette smoke for 3 weeks to induce pulmonary distress. Weeks 1 and 2consisted of 5 consecutive exposure days, while mice were exposed for 4consecutive days during week 3. Mice were exposed to 5 cigarettes eachday with 24 minute intervals of cigarette exposure (16 minutes) andfresh air (8 minutes). Eighteen hours following the final exposure, micewere lavaged with 2×0.8 ml of Hank's Salt Solution (0.6 mM EDTA). Thesupernatant and cell pellet were collected from the bronchial alveolarlavage following centrifugation for 10 minutes. Total macrophage andneutrophil cell counts in the bronchial alveolar lavage for eachexposure group are shown in Table 20.

TABLE 20 Total Cells Cell Counts × 10{circumflex over ( )}5 Mouse meanSD N SEM WT 2.21 1.47 9 0.49 IL1RL2 KO 2.45 0.87 6 0.36 WT + CS 9.072.83 10 0.90 IL-1RL2 KO + CS 5.32 1.03 10 0.32 Macrophages Cell Counts ×10{circumflex over ( )}5 Mouse mean SD N SEM WT 2.08 1.62 9 0.54 IL1RL2KO 2.40 0.86 6 0.35 WT + CS 3.36 1.46 10 0.46 IL-1RL2 KO + CS 3.22 0.8610 0.27

1. An anti-IL-36R antibody or antigen-binding fragment thereof, whichbinds to human IL-36R at a K_(D) equal to or <0.1 nM.
 2. An anti-IL-36Rantibody or antigen-binding fragment thereof according to claim 1,wherein the said antibody or antigen-binding fragment is a monoclonalantibody or antigen-binding fragment thereof.
 3. An anti-IL-36R antibodyor antigen-binding fragment thereof according to claim 1, wherein thesaid antibody or antigen-binding fragment is a humanized antibody orantigen-binding fragment thereof.
 4. An anti-IL-36R antibody orantigen-binding fragment thereof according to claim 3, which binds tohuman IL-36R at a K_(D) equal to or <50 pM.
 5. An anti-IL-36R antibodyor antigen-binding fragment thereof according to claim 1, which does notbind to human IL-1R1. 6-15. (canceled)
 16. An anti-IL-36R antibody orantigen-binding fragment thereof according to claim 1, wherein theantibody or antigen-binding fragment fragment thereof comprises: a lightchain variable region comprising the amino acid sequence of SEQ ID NO:27 (L-CDR1); the amino acid sequence of SEQ ID NO: 36 (L-CDR2); theamino acid sequence of SEQ ID NO: 45 (L-CDR3); and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 107(H-CDR1); the amino acid sequence of SEQ ID NO: 63 (H-CDR2); the aminoacid sequence of SEQ ID NO: 73 (H-CDR3); or a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 27 (L-CDR1); theamino acid sequence of SEQ ID NO: 36 (L-CDR2); the amino acid sequenceof SEQ ID NO: 45 (L-CDR3); and a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 107 (H-CDR1); the amino acidsequence of SEQ ID NO: 64 (H-CDR2); the amino acid sequence of SEQ IDNO: 73 (H-CDR3); or a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 27 (L-CDR1); the amino acid sequence of SEQID NO: 36 (L-CDR2); the amino acid sequence of SEQ ID NO: 45 (L-CDR3);and a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 54 (H-CDR1); the amino acid sequence of SEQ ID NO: 63 or 64(H-CDR2); the amino acid sequence of SEQ ID NO: 73 (H-CDR3).
 17. Ananti-IL-36R antibody or antigen-binding fragment thereof according toclaim 1, wherein the antibody or antigen-binding fragment fragmentthereof comprises a light chain variable region comprising the aminoacid sequence of any one of SEQ ID NO: 84, 85 or 86; and a heavy chainvariable region comprising the amino acid sequence of any one of SEQ IDNO: 96, 97, 98, 99, 100 or
 101. 18. An anti-IL-36R antibody orantigen-binding fragment thereof according to claim 17, wherein theantibody or antigen-binding fragment fragment thereof comprises a lightchain variable region comprising the amino acid sequence of SEQ ID NO:85; and a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 100; or a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 85; and a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:101.
 19. An anti-IL-36Rantibody or antigen-binding fragment thereof according to claim 17,wherein the antibody or antigen-binding fragment fragment thereofcomprises a light chain variable region comprising the amino acidsequence of SEQ ID NO: 86; and a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 100; or a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 86; and a heavychain variable region comprising the amino acid sequence of SEQ IDNO:101. 20-26. (canceled)
 27. An anti-IL-36R antibody according to claim1, wherein the antibody comprises a light chain comprising the aminoacid sequence of any one of SEQ ID NO: 122, 123 or 124; and a heavychain comprising the amino acid sequence of any one of SEQ ID NO: 134,135, 136, 137, 138 or
 139. 28. An anti-IL-36R antibody according toclaim 27, wherein the antibody comprises a light chain comprising theamino acid sequence of SEQ ID NO: 123; and a heavy chain comprising theamino acid sequence of SEQ ID NO:
 138. 29. An anti-IL-36R antibodyaccording to claim 27, wherein the antibody comprises a light chaincomprising the amino acid sequence of SEQ ID NO: 123; and a heavy chaincomprising the amino acid sequence of SEQ ID NO:
 139. 30. An anti-IL-36Rantibody according to claim 27, wherein the antibody comprises a lightchain comprising the amino acid sequence of SEQ ID NO: 124; and a heavychain comprising the amino acid sequence of SEQ ID NO:
 138. 31-32.(canceled)
 33. An anti-IL-36R antibody or antigen-binding fragmentthereof according to claim 1, wherein the antibody or antigen-bindingfragment fragment thereof comprises: a) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 27 (L-CDR1); the aminoacid sequence of SEQ ID NO: 36(L-CDR2); the amino acid sequence of SEQID NO: 45 (L-CDR3); and b) a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 107 (H-CDR1); the amino acid sequenceof SEQ ID NO: 63 (H-CDR2); the amino acid sequence of SEQ ID NO: 73(H-CDR3).
 34. An anti-IL-36R antibody or antigen-binding fragmentthereof according to claim 1, wherein the antibody or antigen-bindingfragment fragment thereof comprises: a) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 27 (L-CDR1); the aminoacid sequence of SEQ ID NO: 36(L-CDR2); the amino acid sequence of SEQID NO: 45 (L-CDR3); and b) a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 107 (H-CDR1); the amino acid sequenceof SEQ ID NO: 64 (H-CDR2); the amino acid sequence of SEQ ID NO: 73(H-CDR3).
 35. An anti-IL-36R antibody or antigen-binding fragmentthereof according to claim 1, wherein the antibody or antigen-bindingfragment fragment thereof comprises: a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 21 (L-CDR1); the aminoacid sequence of SEQ ID NO: 30 (L-CDR2); the amino acid sequence of SEQID NO: 39 (L-CDR3); and a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 48 (H-CDR1); the amino acid sequenceof SEQ ID NO: 57 (H-CDR2); the amino acid sequence of SEQ ID NO: 67(H-CDR3); or a light chain variable region comprising the amino acidsequence of SEQ ID NO: 22 (L-CDR1); the amino acid sequence of SEQ IDNO: 31 (L-CDR2); the amino acid sequence of SEQ ID NO: 40 (L-CDR3); anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 49 (H-CDR1); the amino acid sequence of SEQ ID NO: 58 (H-CDR2);the amino acid sequence of SEQ ID NO: 68 (H-CDR3); or a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 23(L-CDR1); the amino acid sequence of SEQ ID NO: 32 (L-CDR2); the aminoacid sequence of SEQ ID NO: 41 (L-CDR3); and a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 50 (H-CDR1); theamino acid sequence of SEQ ID NO: 59 (H-CDR2); the amino acid sequenceof SEQ ID NO: 69 (H-CDR3); or a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 24 (L-CDR1); the amino acidsequence of SEQ ID NO: 33 (L-CDR2); the amino acid sequence of SEQ IDNO: 42 (L-CDR3); and a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 51 (H-CDR1); the amino acid sequence of SEQID NO: 60 (H-CDR2); the amino acid sequence of SEQ ID NO: 70 (H-CDR3);or a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 25 (L-CDR1); the amino acid sequence of SEQ ID NO: 34(L-CDR2); the amino acid sequence of SEQ ID NO: 43 (L-CDR3); and a heavychain variable region comprising the amino acid sequence of SEQ ID NO:52 (H-CDR1); the amino acid sequence of SEQ ID NO: 61 (H-CDR2); theamino acid sequence of SEQ ID NO: 71 (H-CDR3); or a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 26 (L-CDR1); theamino acid sequence of SEQ ID NO: 35 (L-CDR2); the amino acid sequenceof SEQ ID NO: 44 (L-CDR3); and a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 53 (H-CDR1); the amino acidsequence of SEQ ID NO: 62 (H-CDR2); the amino acid sequence of SEQ IDNO: 72 (H-CDR3); or a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 27 (L-CDR1); the amino acid sequence of SEQID NO: 36 (L-CDR2); the amino acid sequence of SEQ ID NO: 45 (L-CDR3);and a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 54 (H-CDR1); the amino acid sequence of SEQ ID NO: 63(H-CDR2); the amino acid sequence of SEQ ID NO: 73 (H-CDR3); or a lightchain variable region comprising the amino acid sequence of SEQ ID NO:27 (L-CDR1); the amino acid sequence of SEQ ID NO: 36 (L-CDR2); theamino acid sequence of SEQ ID NO: 45 (L-CDR3); and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 54(H-CDR1); the amino acid sequence of SEQ ID NO: 64 (H-CDR2); the aminoacid sequence of SEQ ID NO: 73 (H-CDR3); or a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 28 (L-CDR1); theamino acid sequence of SEQ ID NO: 37 (L-CDR2); the amino acid sequenceof SEQ ID NO: 46 (L-CDR3); and a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 55 (H-CDR1); the amino acidsequence of SEQ ID NO: 65 (H-CDR2); the amino acid sequence of SEQ IDNO: 74 (H-CDR3); or a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 29 (L-CDR1); the amino acid sequence of SEQID NO: 38 (L-CDR2); the amino acid sequence of SEQ ID NO: 47 (L-CDR3);and a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 56 (H-CDR1); the amino acid sequence of SEQ ID NO: 66(H-CDR2); the amino acid sequence of SEQ ID NO: 75 (H-CDR3).
 36. Ananti-IL-36R antibody or antigen-binding fragment thereof according toclaim 1, wherein the antibody or antigen-binding fragment fragmentthereof comprises a light chain protein comprising the amino acidsequence of SEQ ID NO: 1; and a heavy chain protein comprising the aminoacid sequence of SEQ ID NO: 11; or a light chain protein comprising theamino acid sequence of SEQ ID NO: 2; and a heavy chain proteincomprising the amino acid sequence of SEQ ID NO: 12; or a light chainprotein comprising the amino acid sequence of SEQ ID NO: 3; and a heavychain protein comprising the amino acid sequence of SEQ ID NO: 13; or alight chain protein comprising the amino acid sequence of SEQ ID NO: 4;and a heavy chain protein comprising the amino acid sequence of SEQ IDNO: 14; or a light chain protein comprising the amino acid sequence ofSEQ ID NO: 5; and a heavy chain protein comprising the amino acidsequence of SEQ ID NO: 15; or a light chain protein comprising the aminoacid sequence of SEQ ID NO: 6; and a heavy chain protein comprising theamino acid sequence of SEQ ID NO: 16; or a light chain proteincomprising the amino acid sequence of SEQ ID NO: 7; and a heavy chainprotein comprising the amino acid sequence of SEQ ID NO: 17; or a lightchain protein comprising the amino acid sequence of SEQ ID NO: 8; and aheavy chain protein comprising the amino acid sequence of SEQ ID NO: 18;or a light chain protein comprising the amino acid sequence of SEQ IDNO: 9; and a heavy chain protein comprising the amino acid sequence ofSEQ ID NO: 19; or a light chain protein comprising the amino acidsequence of SEQ ID NO: 10; and a heavy chain protein comprising theamino acid sequence of SEQ ID NO:
 20. 37. A pharmaceutical compositioncomprising an antibody or antigen-binding fragment according to claim 1and a pharmaceutically acceptable carrier.
 38. A method of treating adisease comprising administering the antibody or antigen-bindingfragment according to claim 1 or a pharmaceutical composition thereof,to a patient in need thereof, wherein the disease is selected from aninflammatory disease, an autoimmune disease, a respiratory disease, ametabolic disorder, an epithelial mediated inflammatory disorder,fibrosis and cancer.
 39. A method according to claim 38, wherein thedisease is selected from psoriasis, inflammatory bowel disease,psoriatic arthritis, multiple sclerosis, rheumatoid arthritis, COPD,chronic asthma and ankylosing spondylitis.
 40. A method according toclaim 38, wherein the disease is Crohn's disease.
 41. An isolatedpolynucleotide according to claim 1, encoding a sequence as defined byone or more of SEQ ID NOs. 1 to
 140. 42. A vector comprising apolynucleotide according to claim 41, preferably an expression vector,more preferred a vector comprising the polynucleotide according to theinvention in functional association with an expression control sequence.43. A host cell comprising a polynucleotide according to claim 41, and avector.
 44. Method for the production of an anti-IL-36R antibody orantigen-binding fragment according to one claim 1 comprising the steps(a) cultivating the host cell under conditions allowing the expressionof the anti-IL-36R antibody or antigen-binding fragment and (b)recovering the anti-IL-36R antibody or antigen-binding fragment. 45.Diagnostic kit or diagnostic method comprising an anti-IL-36R antibodyor antigen-binding fragment according to claim 1, or the use thereof.46. Diagnostic kit or diagnostic method according to claim 45, for thediagnosis of an inflammatory disease, an autoimmune disease, arespiratory disease, a metabolic disorder, an epithelial mediatedinflammatory disorder, fibrosis, cancer, psoriasis, inflammatory boweldisease, psoriatic arthritis, multiple sclerosis, rheumatoid arthritis,COPD, chronic asthma, ankylosing spondylitis, or Crohn's disease.